Global gene regulators (ggr) as vaccine candidates against paratuberculosis

ABSTRACT

Described herein is a mycobacterium mutant, comprising at least one mutation in at least one gene sequence encoding global gene regulators (GGRs) selected from the group consisting of sigH, sigL, sigE, ECF-1, and mixtures thereof, wherein the GGR gene is at least partially inactivated. Described herein also is a vaccine based on the mutant and a method of differentiating between subjects that have been infected with mycobacterium and subjects that have not been infected with mycobacterium or have been vaccinated with a mycobacterium vaccine.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. patent application Ser. No.15/492,553, filed Apr. 20, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/193,818 filed Feb. 28, 2014, which claimspriority to U.S. Patent Application 61/777,907, filed Mar. 12, 2013,each of which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under 14-CRHF-0-6055awarded by the USDA/NIFA. The government has certain rights in theinvention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named“960296_01676_ST25.txt” which is 6.05 kb in size was created on Feb. 28,2014 and electronically submitted via EFS-Web herewith the applicationis incorporated herein by reference in its entirety.

BACKGROUND

Mycobacterium avium subspecies paratuberculosis (a.k.a. M.paratuberculosis) is the etiological agent of Johne's disease, a chronicenteritis of domestic and wild animals, especially ruminants. Johne'sdisease (JD) has been reported on every continent (1-3), and isconsidered one of the greatest causes of economic hardship to theruminant industries (4). More than two thirds of the U.S. dairy herdsare infected with JD (5) and this wide distribution of the disease,reduced milk production and premature culling of infected animalstogether causes severe economic losses estimated to be over $200 milliona year for the dairy industry (6,7).

Majority of the M. paratuberculosis infection occurs through fecal-oralroute and the mycobacteria are endocytosed by enterocytes and M cells inthe Peyer's patches of the ileum (8,9). After subsequent internalizationby subepithelial and intraepithelial macrophages, M. paratuberculosis isable to survive and persist within the cells (10) using mechanisms thatare not completely understood. Several studies examined gene expressionpatterns and host defense mechanisms of bovine macrophages fromnaturally infected cows (11), peripheral blood mononuclear cells (PBMC)(12) or monocytes-derived macrophages (MDMs) (13,14) following infectionwith M. paratuberculosis. Alternatively, our group characterized thegeneral and specific stress responses of M. paratuberculosis undervarious in vitro conditions as well as the transcriptomes of M.paratuberculosis in fecal samples from diseased cows (15).

Survival of M. paratuberculosis in environmental samples (16),macrophages (17) and animal models (18,19) is well-documented, however,the genetic basis for this survival remains unknown. Reports employing alarge-scale screening of M. paratuberculosis mutants in relevant animalmodels (20,21) provided some insights into virulence of this organismwith the identification of novel virulence factors associated withbiofilm formation (22) and epithelial cell invasion (23). Recently, Zhu,et al. analyzed intracellular M. paratuberculosis gene expressionpatterns in bovine MDMs using SCOTS (selective capture of transcribedsequences), identifying similar patterns of responses to oxidativestress, metabolic activity, and cell survival among M. paratuberculosiswith distinct host origins (24). The same group further analyzed theexpression profiles of M. paratuberculosis isolated from naturallyinfected bovine tissues, identifying tissue-specific pathways (25).However, no comprehensive study has been conducted to clarify therelationship between M. paratuberculosis gene expression and specifichost microenvironments following macrophage infection.

The current vaccine has a limited use to farmers in some regions (e.g. afew European countries) because of its inability to reduce M. apshedding in feces of infected animals, the main source for spreading JD.There is only one vaccine (Mycopar®, Boehringer Ingelheim) approved forlimited use in the USA. This vaccine causes significant granulomaformation at the site of inoculation 21, which persists throughout theanimal's life, increasing the possibility for tissue condemnation at theslaughterhouse.

Despite the ability of this vaccine to induce cell mediated immunity inanimals¹⁷, shedding of M. ap from vaccinated animals continues to causea problem for transmitting the disease to naïve animals (5,22). Insheep, some animals both shed M. ap and died from multi-bacillary formof JD despite being vaccinated (23). In a long-term study of the effectof killed vaccine on dairy herds to reduce the transmission of thedisease, no significant difference in prevalence was found betweenvaccinated and non-vaccinated herds (22). In another study of commercialJD vaccines, cross reactivity to bovine tuberculosis was prominent,further hampering efforts for controlling tuberculosis in farm animals(24,25). More efforts are needed to better understand the pathogenesisof JD and to plan an effective control strategy.

The present invention starts with a goal to gain insights into how M.paratuberculosis respond to the intracellular microenvironments ofmacrophages, the primary site for mycobacterial persistence within thehost, using targeted mutagenesis and an array of transcriptome analyses.In this study, the inventors took advantage of analytical microscopy todefine the phagosome environment of M. paratuberculosis-containingmacrophages in association with the expression profile of mycobacterialbacilli using DNA microarrays. The analysis suggested key changes in themetabolic pathways of M. paratuberculosis once the bacteria encounteractive macrophages and the activation of various alternative sigmafactors (Global Gene Regulators, GGRs) that could help M.paratuberculosis survive the hostile intracellular environment ofmacrophages. One such alternative GGR, sigH, has been shown tocontribute to the resistance encountered during variable environmentalstress conditions, such as temperature and oxidative stress in M.tuberculosis (26,27). However, the basis of transcriptional regulationof sigH remains elusive in M. paratuberculosis.

The inventors therefore sought to define the gene regulatory networkunder control of GGRs, for example, sigH, sigL, sigE and ECF-1 in M.paratuberculosis. Accordingly, they confirmed a role for these key GGRsactivated inside macrophages in defending M. paratuberculosis againstthiol-specific oxidative stress and characterized the effect of theseGGRs on global transcriptome in M. paratuberculosis. Based on theresults, the inventor envisions that the GGR mutants could play animportant role in designing effective vaccines against mycobacterialinfections.

BRIEF SUMMARY OF THE INVENTION

In its first aspect, the present invention relates to a mycobacteriummutant, comprising at least one mutation in at least one gene sequenceencoding global gene regulators (GGRs) selected from the groupconsisting of sigH, sigL, sigE, ECF-1, and mixtures thereof, wherein theGGR gene is at least partially inactivated.

In some embodiments, the mycobacterium is selected from the groupconsisting of Mycobacterium avium subspecies paratuberculosis (M. ap),Mycobacterium bovis (M. bovis), Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium avium subsp. avium (M. avium), and mixturesthereof. Preferably, the mycobacterium is M. ap, M. bovis, or M.tuberculosis.

In some embodiments, the GGR is M. ap sigH (SEQ ID NO:1) or a sequencesubstantially identical to SEQ ID NO:1. In some embodiments, the GGR isM. ap sigL (SEQ ID NO:2) or a sequence substantially identical to SEQ IDNO:2. In some embodiments, the GGR is M. ap sigE (SEQ ID NO:3) or asequence substantially identical to SEQ ID NO:3. In some embodiments,the GGR is M. ap ECF-1 (SEQ ID NO:4) or a sequence substantiallyidentical to SEQ ID NO:4. In other embodiments, the GGR is M. ap sigB(SEQ ID NO:5) or a sequence substantially identical to SEQ ID NO:5.

In some embodiments, the inactivation of the GGR genes is achieved by atleast partial deletion of the gene sequence encoding GGRs. Preferably,the inactivation is achieved by a complete deletion of the gene sequenceencoding GGRs.

In some embodiments, the inactivation of the GGR genes is achieved by atleast partial transposon insertion of the gene sequence encoding GGRs.Preferably, the inactivation is achieved by a complete transposoninsertion of the gene sequence encoding GGRs.

In some embodiments, the inactivation of the GGR genes is achieved by atleast partial anti-sense construct of the gene sequence encoding GGRs.Preferably, the inactivation is achieved by a complete anti-senseconstruct of the gene sequence encoding GGRs.

In its second aspect, the present invention relates to an isolatedmycobacterial organism comprising a mycobacterium mutant describedherein.

In its third aspect, the present invention relates a mycobacteriumvaccine comprising a mycobacterium mutant described herein. In someembodiments, the vaccine may also comprise the mycobacterium organismdescribed above.

In some embodiments, the vaccine is physically inactivated. Preferably,the vaccine is heat inactivated.

In some embodiments, the vaccine is chemically inactivated. Preferably,the vaccine is formaldehyde inactivated.

In other embodiments, the vaccine is a live attenuated vaccine.

In its fourth aspect, the present invention relates to a method ofdifferentiating between subjects that have been infected withmycobacterium and subjects that have not been infected withmycobacterium or have been vaccinated with a mycobacterium vaccine.

In one embodiment, the method comprises the steps of identifying asequence that is specific to a mycobacterium mutant and is not found inthe wild-type mycobacterium strain; and detecting the presence of thesequence in the subject. In another embodiment, the method comprises thesteps of identifying a sequence that is specific to a wild-typemycobacterium strain and is not found in the mycobacterium mutant; anddetecting the presence or the quantity of the sequence in the subject.

In a preferred embodiment, the mycobacterium mutant is a mutantdescribed herein. In another preferred embodiment, the detection is madeby using probes or primers complementary to the mycobacterium mutant inan amplification reaction. More preferably, the amplification is theloop-mediated isothermal amplification (LAMP).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Temporal expression of M. avium subsp. paratuberculosis sigmafactors within macrophages using DNA microarrays. A selected list of theM. avium subsp. paratuberculosis sigma factor genes are shown undernaïve macrophages. Note that sigH and other ECF-1 (ECF-1 on the chartfor clarity) were upregulated immediately after infection, followed byexpression of sigE and sigB.

FIG. 1B. Temporal expression of M. avium subsp. Paratuberculosis sigmafactors within macrophages using DNA microarrays. A selected list of theM. avium subsp. paratuberculosis sigma factor genes are shown underactivated macrophages. Note that sigH and other ECF-1 (ECF-1 on thechart for clarity) were upregulated immediately after infection,followed by expression of sigE aznd sigB.

FIG. 2A. Survival of M. avium subsp. paratuberculosis strains in bovinemacrophages. Naive MDM cells were infected with ΔsigH mutant andwild-type M. avium subsp. paratuberculosis strains. Cells were lysed at1, 4, and 8 days postinfection, and numbers of viable bacilli weredetermined by CFU plating. The survival levels at 4 and 8 days wererelative to the viable counts of bacterial strains at day 1. Survivaldata represent the average of macrophage infections collected from threedifferent donor animals with significance levels in Student's t test (*,P<0.05). Error bars represent the standard deviations.

FIG. 2B. Survival of M. avium subsp. paratuberculosis strains in bovinemacrophages. IFN-γ-pretreated MDM cells were infected with ΔsigH mutantand wild-type M. avium subsp. paratuberculosis strains. Cells were lysedat 1, 4, and 8 days postinfection, and numbers of viable bacilli weredetermined by CFU plating. The survival levels at 4 and 8 days wererelative to the viable counts of bacterial strains at day 1. Survivaldata represent the average of macrophage infections collected from threedifferent donor animals with significance levels in Student's t test (*,P<0.05). Error bars represent the standard deviations.

FIG. 3A. Virulence of M. avium subsp. paratuberculosis K10 and the ΔsigHmutant in mice. Mice groups (n=15) were inoculated with ˜2×10⁸ CFU/mouseof M. avium subsp. paratuberculosis wild-type strain or the ΔsigH mutantvia intraperitoneal injection. Intestines were collected at 3, 6, and 12wpi (n=5 mice/group/time point) and cultured for bacterial counts.Colony counts for each group are represented by scattered plotsaccompanied with a median line. Organs with significant difference inbacterial load were denoted with * for P values of <0.05 or ** for Pvalues of <0.01).

FIG. 3B. Virulence of M. avium subsp. paratuberculosis K10 and the ΔsigHmutant in mice. Mice groups (n=15) were inoculated with ˜2×10⁸ CFU/mouseof M. avium subsp. paratuberculosis wild-type strain or the ΔsigH mutantvia intraperitoneal injection. Spleens were collected at 3, 6, and 12wpi (n=5 mice/group/time point) and cultured for bacterial counts.Colony counts for each group are represented by scattered plotsaccompanied with a median line. Organs with significant difference inbacterial load were denoted with * for P values of <0.05 or ** for Pvalues of <0.01).

FIG. 3C. Pathology of spleen collected from mice infected with M. aviumsubsp. paratuberculosis K10. H&E-stained sections with 100×magnification (scale bar=200 μm) are shown. Inset images (1,000xmagnification, scale bar=10 μm) show the M. avium subsp.paratuberculosis bacilli in purple color (arrows). WP, white pulp; RP,red pulp.

FIG. 3D. Pathology of spleen collected from mice infected with M. aviumsubsp. paratuberculosis K10 and its isogenic ΔsigL mutant. H&E-stainedsections with 100× magnification (scale bar=200 μm) are shown. Insetimages (1,000× magnification, scale bar=10 μm) show the M. avium subsp.paratuberculosis bacilli in purple color (arrows). WP, white pulp; RP,red pulp.

FIG. 4A. Construction of M. avium subsp. paratuberculosis ΔsigL usingwild-type M. avium subsp. paratuberculosis strain. A Physical mapdisplaying the deletion of sigL (MAP4201) gene with homologousrecombination via pYUB854 cosmid shuttle cloning vector, which resultedin the deletion of ˜750 bp coding region and the insertion of a ˜2 kbregion encoding a hygromycin resistance cassette.

FIG. 4B. Construction of M. avium subsp. paratuberculosis ΔsigL usingwild-type M. avium subsp. paratuberculosis strain. The M. avium subsp.paratuberculosis ΔsigL mutant was confirmed with PCR and sequenceverification using genomic DNA from the wild-type and the mutantstrains. Primer pairs were designed for the sigL region, hygromycinresistance gene (hyg^(r)), or the recombinant region after allelicexchange. A 1.5% agarose gel showed amplicons from the sigL region onlywhen wild-type genomic DNA was used (lane 1), whereas hyg^(r) wasamplified only from the M. avium subsp. paratuberculosis ΔsigL mutantgenomic DNA (lane 4). Lane 6 showed amplicon from the recombinant regiononly when M. avium subsp. paratuberculosis ΔsigL mutant genomic DNA wasused.

FIG. 4C. Construction of M. avium subsp. paratuberculosis ΔsigL usingwild-type M. avium subsp. paratuberculosis strain. The polarity of theM. avium subsp. paratuberculosis ΔsigL knockout mutant was assessedusing reverse-transcriptase PCR analysis to check for transcription ofits neighboring genes. In the wild-type (left) and complemented (right)strains, positive bands show that map', sigL and the downstream geneMAP4202 are both encoded in the genome and transcribed (amplified fromcDNA), with no amplification from RNA used as a negative control. In theM. avium subsp. paratuberculosis ΔsigL mutant (middle), the sigL codingregion is absent in the genome or as cDNA, but transcripts for theneighboring genes are present.

FIG. 5A. Analysis of immune responses in immunized mice beforechallenge. Scheme illustrating the immunization study. C57BL/6 micereceived a total of 2 doses, each containing ˜2×10⁶ CFU M. avium subsp.paratuberculosis ΔsigL by s.c injection. Mock group received PBS buffer.Following vaccination, both groups of mice were challenged withwild-type M. avium subsp. paratuberculosis strain as described above.After 6 weeks post-immunization (6PWI; week° in the scheme) or 6 weekspost-challenge (WPC), mice (N=4-6) from each group were sacrificed foranalysis of immune response.

FIG. 5B. Analysis of immune responses in immunized mice beforechallenge. Splenocytes (6PWI) were isolated and re-stimulated in vitrowith Johnin PPD to measure IFN-γ levels from culture supernatant byELISA after 48 h.

FIG. 5C. Analysis of immune responses in immunized mice beforechallenge. M. avium subsp. paratuberculosis specific antibody (anti-PPDjantibodies) in the mouse sera (6PWI) was detected by ELISA (OD 450nm)using Horseradish peroxidase conjugated rabbit anti-mouse antibody.*p<0.05.

FIG. 6A. Protective efficacy of immunization. At 6 weeks followingvaccination, mice received a challenge dose containing ˜7×10⁸ CFUwild-type M. avium subsp. paratuberculosis strain i.p (see FIG. 5A).Following challenge, mice groups (N=4) were sacrificed at 6 and 12 weeksand bacterial burden was analyzed in the spleen.

FIG. 6B. Protective efficacy of immunization. At 6 weeks followingvaccination, mice received a challenge dose containing ˜7×10⁸ CFUwild-type M. avium subsp. paratuberculosis strain i.p (see FIG. 5A).Following challenge, mice groups (N=4) were sacrificed at 6 and 12 weeksand bacterial burden was analyzed in the liver.

FIG. 6C. Protective efficacy of immunization. At 6 weeks followingvaccination, mice received a challenge dose containing ˜7×10⁸ CFUwild-type M. avium subsp. paratuberculosis strain i.p (see FIG. 5A).Following challenge, mice groups (N=4) were sacrificed at 6 and 12 weeksand bacterial burden was analyzed in the intestine. Horizontal linesindicate median value. Statistical analyses were done using student's ttest and Mann-Whitney test to evaluate differences in bacterial organload among mice groups vaccinated with the PBS (mock) or M. avium subsp.paratuberculosis ΔsigL mutant.

FIG. 6D. Secretion of IFN-γ production (6WPC) from the cell supernatantwas measured by ELISA. The histograms show mean values with error barsrepresenting the standard deviation. *p<0.05, **p<0.01.

FIG. 7A. Pathological analysis of mice organs following vaccination.Photographs shows haematoxylin and eosin staining liver.

FIG. 7B. Pathological analysis of mice organs following vaccination.Photographs shows haematoxylin and eosin staining liver.

FIG. 7C. Pathological analysis of mice organs following vaccination.Photographs shows haematoxylin and eosin staining intestine section(100× magnification, scale bar=200 μm) from mock and M. avium subsp.paratuberculosis ΔsigL vaccinated animals following challenge withwild-type M. avium subsp. paratuberculosis strain at 6WPC and 12WPC.Ziehl-Neelsen staining of both liver and intestine displayed higheracid-fast bacilli (inset images; 1000× magnification, scale bar=10 μm)in the mock vaccinated animals compared to the ones that received M.avium subsp. paratuberculosis ΔsigL vaccination.

FIG. 7D. Pathological analysis of mice organs following vaccination.Photographs shows haematoxylin and eosin staining intestine section(100× magnification, scale bar=200 μm) from mock and M. avium subsp.paratuberculosis ΔsigL vaccinated animals following challenge withwild-type M. avium subsp. paratuberculosis strain at 6WPC and 12WPC.Ziehl-Neelsen staining of both liver and intestine displayed higheracid-fast bacilli (inset images; 1000× magnification, scale bar=10 μm)in the mock vaccinated animals compared to the ones that received M.avium subsp. paratuberculosis ΔsigL vaccination.

FIG. 8A. Virulence of M. ap mutants in BALB/c mice (n=15/group)following IP. Spleen (circle).

FIG. 8B. Virulence of M. ap mutants in BALB/c mice (n=15/group)following IP. Liver (square) were collected at 3, 6, and 12 WPI.Colonization levels were compared to M. ap K10 (Continued line). *denotes significant difference between groups in Student's t-test(p<0.05).

FIG. 9A. Pathology of spleen collected from mice infected with wild typeM. ap.

FIG. 9B. Pathology of spleen collected from mice infected with isogenicmutant ΔsigH. H&E stained sections with 100× (Bar=200 μm) are shown.Inset images (1000×, Bar=10 μm). WP, white pulp; RP, red pulp.

FIG. 10A. Vaccine potential of M. ap σ factors deleted mutants. Recoveryof virulent M. ap (cfu/g tissue) from Intestine. of mice immunized withmock (PBS), ΔsigL or ΔsigH at 6 and 12 weeks following challenge. Colonyforming unit (cfu) counts are shown as a scatter plot where the barrepresents the median. *p<0.05.

FIG. 10B. Vaccine potential of M. ap σ factors deleted mutants. Recoveryof virulent M. ap (cfu/g tissue) from Liver of mice immunized with mock(PBS), ΔsigL or ΔsigH at 6 and 12 weeks following challenge. Colonyforming unit (cfu) counts are shown as a scatter plot where the barrepresents the median. *p<0.05.

DESCRIPTION OF THE INVENTION

The present invention provides mycobacterium mutants and a vaccine basedon the mutants for successfully generating immune response to theinfection by Mycobacteria, and in particular for generating immuneresponse to infection caused by Mycobacterium avium subspeciesparatuberculosis (M. ap). The present invention is based on theidentification of the gene regulatory network under control of globalgene regulators (GGRs) (e.g. sigma factors, transcriptional regulators).It is found that some of key GGRs can be genetically mutated to beactivated inside macrophages in defending M. paratuberculosis againstthiol-specific oxidative stress and characterized the effect of GGRs onglobal transcriptome in M. paratuberculosis. It is envisioned that amutant with the deletion, inactivation or reduction of GGRs gene couldprovide strains capable of replication in hosts to generate enoughprotective immunity.

Definitions

A “mycobacterium” as used herein, refers to micro-organisms of the genusMycobacterium (sometimes abbreviated as M. herein), from the family ofMycobacteriaceae. The particularly interested mycobacteria for thepurpose of the present invention include members of Mycobacterium aviumsubspecies paratuberculosis (M. ap), mycobacterium bovis (M. bovis),Mycobacterium avium subsp. avium (M. avium), M. bovis BCG (the strainmost often used for vaccination purposes), mycobacterium tuberculosis(M. tuberculosis), which includes the species M. tuberculosis (the majorcause of human tuberculosis), M. africanum, M. microti, M. canetti, andM. pinnipedii.

A “global gene regulator, GGR” refers to any protein needed forinitiation of RNA synthesis, for example, “sigma factors” and“transcriptional regulators”. They are bacterial transcriptioninitiation factors that enable specific binding of RNA polymerase togene promoters. RNA polymerase holoenzyme complex consists of core RNApolymerase and a GGR executes transcription of a DNA template strand.The specific GGR used to initiate transcription of a given gene willvary, depending on the gene and on the environmental signals needed toinitiate transcription of that gene.

A “mutation” as used herein refers to a mutation in the genetic material(typically DNA), in particular a non-naturally occurring mutation,obtained via genetic engineering techniques. Mutations may includeinsertions, deletions, anti-sense constructs, substitutions (e.g.,transitions, transversion), transpositions, inversions and combinationsthereof. Mutations also include mutations upstream of the start codon,such as in the promoter region, particularly mutations within 30nucleotides upstream of the start codon, as long as these mutationsaffect the levels and/or function of the gene product. Mutations mayinvolve only a single nucleotide (e.g., a point mutation or a singlenucleotide polymorphism) or multiple nucleotides.

In some embodiments, the mutation is a partial or complete deletion ofthe gene. In some embodiments, the mutation is introduced by insertingheterologous sequences into the gene of interest. In other embodiments,the mutation is introduced by replacing a portion of the wild-type geneor allele, or a majority of the wild-type gene or allele, with aheterologous sequence, or an engineered (e.g., manually altered,disrupted, or changed), non-functional, copy of the wild-type sequence.In some embodiments, the mutation is introduced by a partial or completeanti-sense construct, which refers to a DNA sequence complementary to aparticular gene. When such construct is introduced in the genome, itwill be transcribed in RNA and will be paired with the RNA of the geneit is complementary to. This complex of two RNA (double stranded RNA)will then be degraded so that no protein of the gene will be expressed.

Preferably, the mutation is done by deleting at least about 10%, atleast about 20%, at least about 30%, at least about 50%, at least about70% or at least 90% of the wild-type gene sequence. In otherembodiments, the mutation is done by deleting the full wild-typesequence. In some other embodiments, the mutation may even include adeletion of more than 100% of the wild-type gene sequence (e.g., thefull wild-type gene sequence may be deleted but also include one or moreheterologous and/or non-functional nucleic acid sequences attachedthereto or inserted therein).

Mutations could be silent, that is, no phenotypic effect of the mutationis detected. On the other hand, mutations may also cause a phenotypicchange. For example, the expression level of the encoded product isaltered, or the encoded product itself is altered. It is also called asthe gene with the genetically engineered mutation encodes a gene productthat has a reduced (knock-down) or absent (knock-out) functionality.This may be either because there is none or less of the gene productpresent than in the corresponding wild type strain, and/or because thegene product is not or only partially functional.

Thus, the mutations may influence expression levels of the gene product,stability of the gene product, encode defunct or nonsense gene products(e.g. by insertion of a stop codon), encode a different gene product, orany combination of these. For example, a mutation may result in adisrupted gene with decreased levels of expression of a gene product(e.g., protein or RNA) as compared to the wild-type strain (e.g., M.ap). A mutation may also result in an expressed protein with activitythat is lower as compared to the activity of the expressed protein fromthe wild-type strain (e.g., M. ap). In some embodiments, the activity ofthe protein is reduced by 10%, 30%, 50%, 70%, 90% or more.

In specific embodiments of the present invention, mutations areconducted in at least one gene sequence encoding global gene regulators(GGRs). Such mutations result in at least partial inactivation of thefunction of the GGR genes. The term “function” as used herein isintended to mean the function of GGR gene sequences directly and/orindirectly attributable to the expression of the GGR genes. The“function” also refers to the function and/or activity of the proteinsencoded by GGR genes as to their indirectly and/or directly attributionto infectious and/or inflammasome activation of mycobacteria.

The term “at least partially inactivated” as used herein, is meant toindicate a loss, interruption, or alteration of GGR genes functiondirectly and/or indirectly attributable to the expression of GGR genes.It is also meant to indicate a loss, interruption, alteration of theinfectious and/or inflammasome activation of the protein productsencoded by GGR genes in mycobacteria. Preferably, “at least partiallyinactivated” relates to a loss of more than 10%, preferably more than30%, more preferably more than 50%, more preferably more than 70%, mostpreferably more than 90% of the function directly and/or indirectlyattributable to the expression of a GGR gene in mycobacteria. The “atleast partially inactivation” may also include a inactivation of thefull (100%) function of a GGR gene.

Partial and/or full inactivation of genes in mycobacteria for thepurpose of the present invention can be achieved by any conventionalmethod known in the art of gene mutation, e.g., recombinant insertion,transposon insertion, replacement, deletion, frameshift, anti-senseconstruct and/or homologous recombination. Preferably, the specificmethods for partially and/or fully inactivating a GGR gene inmycobacteria is to delete more than 10%, preferably more than 30%, morepreferably more than 50%, more preferably more than 70%, most preferablymore than 90% of a GGR gene sequence.

The mechanism thereof is not vital to the invention, but typically mayinvolve reduction or loss of transcription and/or translation of theaffected gene, or only transcription/translation of a dysfunctional geneproduct. Genetically engineered mutations in a gene need not necessarilybe in an exon or open reading frame, they can be in an intron or even beupstream of the start codon, as long as it affects the levels of thefunctional gene product. This can easily be checked, e.g. by Q-PCR forgene transcription levels. Functionality of a gene product can also bechecked, as one of skill in the art would know, e.g. by providing anatural substrate to the affected enzyme. Of course, for the purpose ofthe present invention, the inactivation and the extent of inactivationcan best be determined in direct comparison to a wild-type mycobacteriumor a mycobacterium of the same strain under identical experimentalconditions but comprising a full functional and expressed GGR gene.

The term “contiguous portions of a sequence” as used herein refers to anon-interrupted sequence of nucleic acids or amino acids also occurringin the same order in the sequence referred to. Particularly envisagedare contiguous portions having a length of at least 25%, 50%, 70%, 75%,80% or 90% of the length of the reference sequence, and contiguousportions are typically at least 25 nucleic acids or at least 8 aminoacids.

The term “sequence identity” as used herein refers to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Nucleic acid andprotein sequence identities can be evaluated by using any method knownin the art. For example, the identities can be evaluated by using theBasic Local Alignment Search Tool (“BLAST”). The BLAST programs identityhomologous sequences by identifying similar segments between a queryamino or nucleic acid sequence and a test sequence which is preferablyobtained from protein or nuclei acid sequence database. The BLASTprogram can be used with the default parameters or with modifiedparameters provided by the user.

The term “percentage of sequence identity” is calculated by comparingtwo optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G) or the identical amino acid residue (e.g., Ala,Pro, Ser, Thr, GIy, VaI, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp,GIu, Asn, GIn, Cys and Met) occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 25% sequenceidentity. Alternatively, percent identity can be any integer from 25% to100%. More preferred embodiments include at least: 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a referencesequence using the programs described herein; preferably BLAST usingstandard parameters, as described. These values can be appropriatelyadjusted to determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning and the like.

The term “substantial identity” of amino acid sequences for purposes ofthis invention normally means polypeptide sequence identity of at least40%. Preferred percent identity of polypeptides can be any integer from40% to 100%. More preferred embodiments include at least 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 98.7%, or 99%.

Mycobacterium Mutants

According to a first aspect, a mycobacterium mutant is providedcomprising at least one genetically engineered mutation in at lease onegene sequence encoding global gene regulators (GGRs), wherein the GGRgene is at least partially inactivated.

In some embodiments, the mycobacterium is selected from the groupconsisting of mycobacterium avium subspecies paratuberculosis (M. ap),mycobacterium bovis (M. bovis), mycobacterium tuberculosis (M.tuberculosis), Mycobacterium avium subsp. avium (M. avium), and mixturesthereof. Preferably, the mycobacterium is M. ap.

In preferred embodiments, the GGR gene is selected from the groupconsisting of sigH, sigL, sigE, ECF-1, and mixtures thereof.

To inactivate a GGR gene in mycobacteria, one may partially orcompletely delete the sequence of the GGR gene. For example, one mayinsert heterologous sequences into the GGR gene. One may also replace aportion of the GGR gene or allele, or a majority of the GGR gene orallele, with a heterologous sequence, or an engineered (e.g., manuallyaltered, disrupted, or changed), non-functional, copy of the wild-typesequence. Preferably, the mutation is done by deleting at least about10%, at least about 20%, at least about 30%, at least about 50%, atleast about 70% or at least 90% of the wild-type GGR gene sequence. Morepreferably, the mutation is done by deleting the full GGR sequence. Insome other embodiments, the mutation may even include a deletion of morethan 100% of the wild-type GGR gene sequence (e.g., the full wild-typegene sequence may be deleted but also include one or more heterologousand/or non-functional nucleic acid sequences attached thereto orinserted therein).

While these genes across different species of mycobacteria are expected,not all genes in each species have the identical sequences, locations,or functions. But based on sequence homology, one skilled person wouldbe able to readily identify the corresponding (or equivalent) gene inother species. In a particular embodiment, the mycobacterium is M.paratuberculosis strain which has 19 GGRs, as listed in Table 1 below.

TABLE 1 List of 19 sigma factors (global gene regulators, GGR) in M.paratuberculosis genome. Gene Start End Start End Old locus Revisedlength nucleotide nucleotide nucleotide nucleotide Gene tag* locus tag**(bp) number* number* number** number** sigA MAP2820 MAPK_0948 15303148633 3150162 1062326 1060797 sigB MAP2826 MAPK_0942  999 31532583154256 1057701 1056703 sigC MAP1814 MAPK_1954 1380 1991733 19931122219261 2217882 sigD MAP4275 MAPK_4277  597 4742530 4743126 47453394745935 sigE MAP2557c MAPK_1211  756 2874942 2875697 1335260 1336015sigF MAP3406c MAPK_0362  789 1613298 1614107  428572  429360 sigF-likeMAP1474c MAPK_2294  810 3781604 3782392 2596880 2597689 sigG MAP3621cMAPK_0147 1101 4018733 4019833  191134  192234 sigH MAP3324c MAPK_0444 807 3692929 3693735  517229  518035 sigI MAP0170 MAPK_3598  858  173736 174593 4037249 4036392 sigJ MAP3446c MAPK_0322  888 3825108 3825995 384969  385856 sigL MAP4201 MAPK_4203  546 4670740 4671285 46735504674095 sigM MAP4337 MAPK_4339  588 4818485 4819072 4821294 4821881ECF-1 MAP0946c MAPK_2822  924  975766  976689 3234298 3235221 ECF-2MAP1770c MAPK_1998  936 1932783 1933718 2277276 2278211 ECF-3 MAP2166MAPK_1602  891 2397944 2398834 1813051 1812161 ECF-4 MAP1757c MAPK_2011 630 1919501 1920130 2290864 2291493 ECF-5 MAP4114c MAPK_4116 12634584803 4586065 4588877 4587615 ECF-6 MAP4217 MAPK_4219  540 46880354688574 4690845 4691384 *Annotations according to Li, et al., 2005 (35).**Improved annotations according to the revised genome sequence of M.paratuberculosis (GenBank Accession No. AE016958) (38). Bolded geneswere up-regulated during macrophage survival and are potential vaccinemutants.

In the present invention, one of the preferred GGR genes is sigH. Theterm “sigH” as used herein refers to a gene which encodes a RNApolymerase sigma-H factor. In some embodiments, the sigH is the sigH(SEQ ID NO:1) of M. paratuberculosis. It has a size of 807 base pairs(bp) and is located at positions 517229 to 518035 of the M.paratuberculosis genomic sequence. In one specific embodiment, amycobacterium mutant comprises at least one mutation in M. ap sigH (SEQID NO: 1) or contiguous portions thereof, or sequences at least 90%, atleast 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1 orthe contiguous portions thereof. Preferably, the mutation in sigH is atleast partial deletion of the sigH gene, as characterized as “M.paratuberculosis ΔsigH”. As a result of the deletion, the sigH gene isexpressed at levels lower than the corresponding wild-type gene, or notexpressed at all. Preferably, the expression level of sigH gene is solow as to have no effect or the expressed protein is non-functional.

Another preferred GGR gene is sigL (SEQ ID NO:2). The term “sigL” asused herein refers to a gene which encodes a Sigma-L-dependenttranscriptional regulator. In some embodiments, the sigL is the sigL(SEQ ID NO:2) of M. paratuberculosis. It has a size of 546 base pairs(bp) and is located at positions 4673550 to 4674095 of the M.paratuberculosis genomic sequence. In one specific embodiment, amycobacterium mutant comprises at least one mutation in sigL (SEQ ID NO:2) or contiguous portions thereof, or sequences at least 90%, at least95%, at least 98%, or at least 99% identical to SEQ ID NO: 2 or thecontiguous portions thereof. Preferably, the mutation in the sigL geneis at least partial deletion of the sigL gene, as characterized as “M.paratuberculosis ΔsigL”. As a result of the deletion, the sigL gene isexpressed at levels lower than the corresponding wild-type gene, or notexpressed at all. Preferably, the expression level of sigL gene is solow as to have no effect or the expressed protein is non-functional.

Another preferred GGR gene is sigE (SEQ ID NO:3). The term “sigE” asused herein refers to a gene which encodes a RNA polymerase sigma-Efactor. In some embodiments, the sigE is the sigE (SEQ ID NO:3) of M.paratuberculosis. It has a size of 756 base pairs (bp) and is located atpositions 1335260 to 1336015 of the M. paratuberculosis genomicsequence. In one specific embodiment, a mycobacterium mutant comprisesat least one mutation in sigE (SEQ ID NO: 3) or contiguous portionsthereof, or sequences at least 90%, at least 95%, at least 98%, or atleast 99% identical to SEQ ID NO: 3 or the contiguous portions thereof.Preferably, the mutation in sigE gene is at least partial deletion ofthe sigE gene, as characterized as “M. paratuberculosis ΔsigE”. As aresult of the deletion, the sigE gene is expressed at levels lower thanthe corresponding wild-type gene, or not expressed at all. Preferably,the expression level of sigE gene is so low as to have no effect or theexpressed protein is non-functional.

Another yet preferred GGR gene is ECF-1. The term “ECF-1” as used hereinrefers to a gene which encodes a RNA polymerase ECF sigma factor. Insome embodiments, the ECF-1 is the ECF-1 (SEQ ID NO:4) of M.paratuberculosis. It has a size of 924 base pairs (bp) and is located atpositions 3234298 to 3235221 of the M. paratuberculosis genomicsequence. In one specific embodiment, a mycobacterium mutant comprisesat least one mutation in ECF-1 (SEQ ID NO: 4) or contiguous portionsthereof, or sequences at least 90%, at least 95%, at least 98%, or atleast 99% identical to SEQ ID NO: 4 or the contiguous portions thereof.Preferably, the mutation in ECF-1 gene is at least partial deletion ofthe ECF-1 gene, as characterized as “M. paratuberculosis Δecf-1”. As aresult of the deletion, the ECF-1 gene is expressed at levels lower thanthe corresponding wild-type gene, or not expressed at all. Preferably,the expression level of ECF-1 gene is so low as to have no effect or theexpressed protein is non-functional.

Another preferred GGR gene is sigB. The term “sigB” as used hereinrefers to a gene which encodes a RNA polymerase sigma-B factor. In someembodiments, the sigB is the sigB (SEQ ID NO:5) of M. paratuberculosis.It has a size of 999 base pairs (bp) and is located at positions 3153258to 3154256 of the M. paratuberculosis genomic sequence. In one specificembodiment, a mycobacterium mutant comprises at least one mutation insigB (SEQ ID NO: 5) or contiguous portions thereof, or sequences atleast 90%, at least 95%, at least 98%, or at least 99% identical to SEQID NO: 5 or the contiguous portions thereof. Preferably, the mutation insigE gene is at least partial deletion of the sigB gene, ascharacterized as “M. paratuberculosis ΔsigB”. As a result of thedeletion, the sigB gene is expressed at levels lower than thecorresponding wild-type gene, or not expressed at all. Preferably, theexpression level of sigB gene is so low as to have no effect or theexpressed protein is non-functional.

Also, in some embodiments, a mycobacterium mutant comprises acombination of two or more of these genes that are mutated. Forinstance, both sigH and sigL genes are mutated, or sigH gene and one ortwo of sigL, sigE, sigB and ECF-1 genes are mutated. Additionally and/oralternatively, one or more of these genes may contain more than onemutation.

In some other embodiments, a mycobacterium mutant may have furthernon-GGR mutations. One example of these further mutations includesadditional mutations in other genes that function independent ordependent from GGRs in directly or indirectly causing mycobacteriainfections. Other non-limiting examples of the further mutation sites,as described in the United States Patent Application Publication Number2007/0134274, include gcpE, pstA, kdpC, papA2, impA, umaA1, fabG2_2,aceAB, mbtH2, 1pqP, map0834c, cspB, 1ipN, map1634 MAP-1, MAP-2, MAP-3,MAP-4, MAP-5, MAP-6, MAP-7, MAP-8, MAP-9, MAP-10, MAP-11, MAP-12,MAP-13, MAP-14, MAP-15, MAP-16, MAP-17, or MAP-18 of M.paratuberculosis, or homologs of these genomic islands, and alsoinclude, as described in the United States Patent ApplicationPublication Number 2007/0134708, MAV-1, MAV-2, MAV-3, MAV-4, MAV-5,MAV-6, MAV-7, MAV-8, MAV-9, MAV-10, MAV-11, MAV-12, MAV-13, MAV-14,MAV-15, MAV-16, MAV-17, MAV-18, MAV-19, MAV-20, MAV-21, MAV-22, MAV-23,or MAV-24, or homologs thereof.

According to the second aspect of the present invention, a mycobacteriummutant may be defined at the protein (amino acid) level. Thus, thepresent invention provides an isolated mycobacterial protein, which isencoded by the gene sequence of a mycobacterium mutant as describedherein. The protein expressed by the mutant may have a lower activityand/or function as compared to the activity of the expressed proteinfrom the wild-type strain (e.g., M. ap). In some embodiments, theactivity and/or the function of the protein is reduced by 10%, 30%, 50%,70%, 90% or more.

Vaccine

The third aspect of the present invention provides a vaccine based onthe mycobacterium mutants described herein. Specifically, in someembodiments, the mycobacterium mutant comprises at least one mutation inat lease one gene sequence encoding global gene regulators (GGRs)described herein, wherein the GGR gene is at least partiallyinactivated. Preferably, the mycobacterium is selected from the groupconsisting of Mycobacterium avium subspecies paratuberculosis (M. ap),Mycobacterium bovis (M. bovis), Mycobacterium tuberculosis (M.tuberculosis), and mixtures thereof. More preferably, the mycobacteriumis M. ap.

The GGR gene may be selected from the group consisting of a sequence ofor sequence substantially identical to sigH (SEQ ID NO:1), sigL (SEQ IDNO:2), sigE (SEQ ID NO:3), ECF-1 (SEQ ID NO:4), sigB (SEQ ID NO:5) andmixtures thereof.

The vaccine of the present invention may be a suspension of liveattenuated, or inactivated (killed) microorganisms comprising themutants described herein. The types microorganisms may vary, such asviruses, bacteria, or rickettsiae of the microorganisms, or of otherantigens such as antigenic proteins and other substances derived fromthem, administered for prevention, amelioration, or treatment ofmycobacterium infections.

In some embodiments, the vaccine is a live attenuated vaccine, whereinthe mycobacterium mutant or the organism thereof used for the inventionis alive. By “alive”, we mean the mutant or the organism thereof iscapable of propagation in a subject, in particular in a mammaliansubject. To be an effective live attenuated vaccine, the livemycobacteria must be attenuated to a degree not harmful to a subject inneed thereof. Hence, the mycobacterium mutant used for the invention ispreferably non-virulent, i.e. the genes responsible for virulence havebeen inactivated, and does not evoke or at least evokes minor diseasesymptoms of a mycobacterial infection in a subject.

In some embodiments, the vaccine is an inactivated vaccine, wherein themycobacterium mutant or the organism thereof used for the invention iskilled or inactivated. The advantage of an inactive vaccine is safety.The vaccine may be inactivated by any art-known method. For example, itmay be inactivated by using chemical methods such as beta-propiolactone,thimerosal or (another mercury donating agent), formaldehyde, etc. Thevaccine may be inactivated by applying physical methods such as heat,UV-light, micro-waves etc. The vaccine may also be inactivated by usingbiological methods such as enzyme-based methods to kill or inactivatethe bacteria and any other method as is commonly applied in the art.

In some other embodiments, the vaccine is an acellular vaccine, i.e., acell-free vaccine prepared from the purified mycobacterial components ofcell-free microorganisms, carrying less risk than whole-cellpreparations.

Vaccination may be accomplished by administering nucleic acid sequenceof the mycobacterium mutant in a form of a DNA vaccine. A “DNA vaccine”or “immunogenic” or “immunological composition” is composed of at leastone vector (e.g., plasmid) which may be expressed by the cellularmachinery of the subject to be vaccinated or inoculated and of apharmaceutically acceptable carrier, vehicle, or excipient. Thenucleotide sequence of this vector encodes one or more immunogens, suchas proteins or glycoproteins capable of inducing, in the subject to bevaccinated or inoculated, a cellular immune response (mobilization ofthe T lymphocytes) and an immune response.

The vaccine of the present invention may be administered to a subject atrisk of being infected by mycobacteria, or to a subject who has beenexposed to mycobacteria, or even to a subject known to be infected withmycobacteria. The typical subject is illustratively a living organismcapable of mounting an immune response to challenge from the vaccine.Non-limiting examples of a subject include a human, any lower primate,cow, camel, dog, cat, rabbit, rat, mouse, guinea pig, pig, hamster,horse, donkey, cattle, opossum, badger, goat, sheep, or other mammals ornon-mammals.

The vaccine of the present invention is suitable for the prophylaxisand/or treatment of a disease or medical condition affected by antigenand/or immunogen expression of the mycobacterium, more preferably theprophylaxis and/or treatment of mycobacterial infections, preferably aninfection of M. ap, M. avium, M. tuberculosis, M. bovis, or mixturesthereof. According to most specific embodiments, the vaccine is avaccine against Johne's disease caused by mycobacterial infection.

The vaccine of the present invention may be administered in anyconventional dosage form in any conventional manner. In someembodiments, routes of administration include, but are not limited to,intravenously, intramuscularly, subcutaneously, intranasally,intrasynovially, by infusion, sublingually, transdermal, orally,topically, or by inhalation. The preferred modes of administration aresubcutaneous, intravenous and intranasal.

In some embodiments, the mycobacterium mutant of the vaccine may beadministered alone or in combination with adjuvants that enhancestability and/or immunogenicity of the mycobacteria, facilitateadministration of pharmaceutical compositions containing them, provideincreased dissolution or dispersion, increase propagative activity,provide adjunct therapy, and the like, including other activeingredients.

In some embodiments, the pharmaceutical dosage forms of the vaccineaccording to the present invention may include pharmaceuticallyacceptable carriers and/or adjuvants known to those of ordinary skill inthe art. These carriers and adjuvants include, for example, ionexchangers, alumina, aluminium stearate, lecithin, serum proteins,buffer substances, water, salts, electrolytes, cellulose-basedsubstances, gelatine, water, petrolatum, animal or vegetable oil,mineral or synthetic oil, saline, dextrose or other sacchari de andglycol compounds such as ethylene glycol, propylene glycol orpolyethylene glycol, antioxidants, lactate, etc. Preferred dosage formsinclude tablets, capsules, solutions, suspensions, emulsions,reconstitutable powders and transdermal patches. Methods for preparingdosage forms are well known in the art.

In other embodiments, the dosage regimen of the vaccine may also bedetermined by the skilled person using his expertise (e.g. singleadministration, repeated administration (twice or more at regular orirregular intervals), etc. This will typically also depend on thedisease to be treated and the subject receiving the treatment. In atypical animal vaccination, a dose of 10⁵-10¹⁰ cfu/animal is given viaparental or oral routes of vaccination. In one embodiment, the dose isin the range of 10⁷-10¹⁰ cfu/animal. In another embodiment, the dose isin the range of 10⁵-10⁹ cfu/animal.

Differentiate Infected from Vaccinated Animals (DIVA)

In its fourth aspect, the present invention provides a method of invitro differentiating between subjects that have been infected withmycobacterium and subjects that have not been infected withmycobacterium or have been vaccinated with a mycobacterium vaccine. Inone embodiment, the method comprises the steps of (a) identifying a genesequence that is specific to a mycobacterium mutant and is not found inthe wild-type mycobacterium strain; and (b) detecting the presence orthe quantity of the sequence in the subject. In other embodiments, themethod comprises the steps of (a) identifying a gene sequence that isspecific to a wild-type mycobacterium strain and is not found in themycobacterium mutant; and (b) detecting the presence or the quantity ofthe sequence in the subject. The mycobacterium mutant for theembodiments is any one mycobacterium mutant described herein. Thesesequences are detectable by use of a complimentary probe or primer.

The gene or the polynucleotides of the invention may contain less thanan entire microbial genome and can be single- or double-stranded nucleicacids. A polynucleotide can be RNA, DNA, cDNA, genomic DNA, chemicallysynthesized RNA or DNA or combinations thereof. The polynucleotides canbe purified free of other components, such as proteins, lipids and otherpolynucleotides. For example, the polynucleotide can be 50%, 75%, 90%,95%, 96%, 97%, 98%, 99%, or 100% purified. The purified polynucleotidescan comprise additional heterologous nucleotides (that is, nucleotidesthat are not from mycobacterium). The purified polynucleotides of theinvention can also comprise other nucleotide sequences, such assequences coding for linkers, primer, signal sequences, TMR stoptransfer sequences, transmembrane domains, or ligands.

The gene or the polynucleotides of the invention can be isolated. Anisolated polynucleotide is a naturally occurring polynucleotide that isnot immediately contiguous with one or both of the 5′ and 3′ flankinggenomic sequences with which it is naturally associated. An isolatedpolynucleotide can be, for example, a recombinant DNA molecule of anylength, provided that the nucleic acid sequences naturally foundimmediately flanking the recombinant DNA molecule in anaturally-occurring genome is removed or absent. Isolatedpolynucleotides also include non-naturally occurring nucleic acidmolecules.

The gene or the polynucleotides of the invention can also comprisefragments that encode immunogenic polypeptides. Polynucleotides of theinvention can encode full-length polypeptides, polypeptide fragments,and variant or fusion polypeptides. Polynucleotides of the invention cancomprise coding sequences for naturally occurring polypeptides or canencode altered sequences that do not occur in nature. If desired,polynucleotides can be cloned into an expression vector comprisingexpression control elements, including for example, origins ofreplication, promoters, enhancers, or other regulatory elements thatdrive expression of the polynucleotides of the invention in host cells.

The gene or the polynucleotides of the invention can be detected by, forexample, a probe or primer, for example, a PCR primer, or can be thebasis for designing a complimentary probe or primer, to detect thepresence and/or quantity of mycobacterium in a subject, such as abiological sample. Probes are molecules capable of interacting with atarget nucleic acid, typically in a sequence specific manner, forexample, through hybridization. Primers are a subset of probes that cansupport an specific enzymatic manipulation and that can hybridize with atarget nucleic acid such that the enzymatic manipulation occurs. Aprimer can be made from any combination of nucleotides or nucleotidederivatives or analogs available in the art that do not interfere withthe enzymatic manipulation. “Specific” means that a gene sequencerecognizes or matches another gene of the invention with greateraffinity than to other non-specific molecules. Preferably, “specificallybinds” or “specific to” also means a gene sequence recognizes andmatches a gene sequence comprised in a wild-type mycobacterium or amycobacterium mutant described herein, with greater affinity than toother non-specific molecules. More preferably, the probe or the primeris complimentary to a mycobacterium mutant with at least one mutation inthe gene of sigH (SEQ ID NO:1), sigL (SEQ ID NO:2), sigE (SEQ ID NO:3),ECF-1 (SEQ ID NO:4), sigB (SEQ ID NO:5) and mixtures thereof.

The hybridization of nucleic acids is well understood in the art.Typically a primer can be made from any combination of nucleotides ornucleotide derivatives or analogs available in the art. The ability ofsuch primers to specifically hybridize to mycobacterium polynucleotidesequences will enable them to be of use in detecting the presence ofcomplementary sequences in a given subject. The primers of the inventioncan hybridize to complementary sequences in a subject such as abiological sample, including, without limitation, saliva, sputum, blood,plasma, serum, urine, feces, cerebrospinal fluid, amniotic fluid, woundexudate, or tissue of the subject. Polynucleotides from the sample canbe, for example, subjected to gel electrophoresis or other sizeseparation techniques or can be immobilized without size separation.

The probes or the primers can also be labeled for the detection.Suitable labels, and methods for labeling primers are known in the art.For example, the label includes, without limitation, radioactive labels,biotin labels, fluorescent labels, chemiluminescent labels,bioluminescent labels, metal chelator labels and enzyme labels. Thepolynucleotides from the sample are contacted with the probes or primersunder hybridization conditions of suitable stringencies. Preferably, theprimer is fluorescent labeled. Also, the detection of the presence orquality of the gene sequence of interest can be accomplished by anymethod known in the art. For instance, the detection can be made by aDNA amplification reaction. In some embodiments, “amplification” of DNAdenotes the use of polymerase chain reaction (PCR) to increase theconcentration of a particular DNA sequence within a mixtures of DNAsequences.

Preferably, the amplification of DNA is done by the loop-mediatedisothermal amplification (LAMP). LAMP was developed by Notomi, et al.(72), incorporated by reference herein in its entirety. Similar to PCR,LAMP utilizes a polymerization-based reaction to amplify DNA fromexamined samples, but the enzyme for LAMP, Bst DNA polymerase largefragment, possesses a DNA strand displacement activity. This makes theDNA extension step possible without having to fully denature DNAtemplates. Moreover, the primers are designed in a way that a hairpinloop structure is formed in the first cycle of amplification, and thefollowing products are further amplified in an auto-cycling manner.Therefore, in about an hour, the repeated reactions can amplify by ˜10⁹copies of DNA molecules and can be done at a constant temperature in asingle heat block, instead of at various cycles of temperature in arelatively expensive thermal cycler.

The detection of LAMP can be made by vary technologies known in the art.Preferably, the detection of LAMP can occur without gel electrophoresisthrough the addition of flourophore (73,74). This detection method couldreadily be used in the field to detect positive samples. As a result, acomplete diagnosis can be done and visualized in about one hour (72).Quantification and multiplexing techniques have also been developedwhich could help determine the bacterial load of a sample (73,74). Inaddition, variants of Bst polymerase and primer additions have increasedBst polymerase stability, decreased amplification time, and provided thepolymerase with the ability to reverse transcribe and amplify RNAtargets (73,74). Specifically, the increase in stability could helptransfer the diagnostic process to the field.

Accordingly, the detection of the presence of the gene or the specificbinding between the gene in mycobacterium mutant and a gene that is nota component of a subject's immune response to a particular vaccine mayindicate a natural or experimental mycobacterium infection. For example,the absence of such binding or presence may indicate the absence ofmycobacterium infection. Or, a second, separate gene, such as a mutatedmycobacterium gene that specific to a component of a subject's immuneresponse to a particular mycobacterium vaccine, may be used to detect acorresponding antibodies produced in response to vaccination. Thus, ifan antibody specific to a gene in mycobacterium vaccine is detected,then the subject has been vaccinated and/or infected. The detection ofneither genes indicates no infection and no vaccination. As such,various combinations can lead to a determination of the vaccinationand/or infection status of the subject.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, useful methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features andadvantages of the invention will be apparent from the detaileddescription and from the claims.

EXAMPLES

The following examples set forth preferred markers and methods inaccordance with the invention. It is to be understood, however, thatthese examples are provided by way of illustration and nothing thereinshould be taken as a limitation upon the overall scope of the invention.

Example 1

The disclosure of Example 1 has been published in Infection andImmnunity, June 2013, 81 (6): 2242-2257, entitled “Key Role for theAlternative Sigma Factor, SigH, in the Intracellular Life ofMycobacterium avium subsp. paratuberculosis during Macrophage Stress,”which is incorporated herein by reference in its entirety.

Bacteria. Escherichia coli DH5α and HB101 were used as host cells forcloning purposes in all experiments presented here. M. avium subsp.paratuberculosis K10 and Mycobacterium smegmatis mc²155 strains weregrown in Middlebrook 7H9 broth and on Middlebrook 7H10 plates aspreviously described (1).

Construction of the ΔsigH mutant. A specialized transduction protocolwas adopted with a few modifications to delete the sigH/MAP3324c geneusing the M. avium subsp. paratuberculosis strain (12). Briefly, two˜900-bp PCR fragments flanking each end of the sigH coding region wereamplified and cloned into the pYUB854 shuttle vector. The resultingpYUB854::sigH allelic-exchange substrate (AES) was then digested withPacI and ligated to the PacI-digested concatemers of atemperature-sensitive phasmid, phAE87. The ligation mixture was thenpackaged into phage particles with an in vitro lambda-packaging system(GIGAPackIII; Stratagene, La Jolla, Calif.). Mid-log-phase Escherichiacoli culture was transduced with the packaged phage particles, resultingin hygromycin-resistant colonies. From the mixture of these colonies,shuttle plasmid DNA was extracted and then electroporated into M.smegmatis competent cells. Lysate of plaques formed at 30° C. from thetransformants was collected, propagated, and titrated in M. smegmatis toproduce a high-titer recombinant phage stock. A mid-log-phase culture ofM. avium subsp. paratuberculosis was transduced with the phage stock atnonpermissive temperature (37° C.) with a multiplicity of infection of10. Individual hygromycin-resistant colonies were picked and grown inbroth medium following gDNA isolation. The genotype of sigH-deletionmutants was confirmed with PCR and sequence analysis as outlined before(12).

Stress treatments of M. avium subsp. paratuberculosis. Wild-type and theΔsigH mutant of M. avium subsp. paratuberculosis were grown to late-logphase (optical density at 600 nm [OD₆₀₀]=1.0), and 200 ul was spread on7H10 agar plates (Difco, Sparks, Md.) supplemented with 0.5% glycerol, 2ug/ml mycobactin J, and 10% ADC (2% glucose, 5% bovine serum albumin[BSA] fraction V, and 0.85% NaCl). For disc diffusion assay (DDA), 20 ulof diamide solution (0.5 M, 1 M, or 1.5 M) and H₂O₂ solution (50 mM, 100mM, or 0.5 M) was impregnated onto each 6-mm disc (Whatman, Piscataway,N.J.), and discs were placed on each of the spread plates. As a positivecontrol, ethambutol discs (5 ug/disc, Sensi-Disc; BD Diagnostics) wereused. Plates were incubated at 37° C. until a thick confluent lawndeveloped. The sustained effect of stressors (diamide and heat shock) onthe viability of the wild type and ΔsigH mutant was monitored bydetermining their CFU counts. Aliquots of M. avium subsp.paratuberculosis cultures 1, 3, and 7 days following continuous exposureto 10 mMdiamide or a 45° C. water bath were serially diluted and plated.In another experiment, M. avium subsp. paratuberculosis cultures frommidlog phase were exposed to 10 mM diamide for 3 h. The cultures werecentrifuged (3,000×g, 4° C., 10 min), and pellets were immediatelystored at −80° C. until RNA extraction.

Mouse infections. For the animal infections with M. avium subsp.paratuberculosis strains, female BALB/c mice (Harlan Laboratories,Indianapolis, Ind.) were purchased at 4 weeks of age and housed in apathogen free environment according to the protocol approved by theInstitutional Animal Care and Use Committee, University ofWisconsin-Madison. Two groups of mice (n=15 per group) were challengedintraperitoneally with the wild-type and ΔsigH mutant strains of M.avium subsp. paratuberculosis. Actual infection inoculum sizes (˜2×10⁸CFU per mouse) of these two strains were similar, as determined by platecount on the day of infection. Mouse groups (n=5) were sacrificed at 3,6, and 12 weeks postinfection (wpi), and samples from livers,intestines, and spleens were collected for bacterial CFU enumeration andhistopathological examinations as described before (17). Portions oflivers, spleens, and intestines were fixed in 10% neutral bufferedformalin before being sectioned and stained with hematoxylin and eosin(H&E) and Ziehl-Neelsen stain. Student's t test and Mann-Whitney testwere used to statistically evaluate differences in CFU counts amongmouse groups infected with the wildtype and ΔsigH mutant strains of M.avium subsp. paratuberculosis.

Bovine blood monocyte isolation and infection. Blood was collected froma Johne's disease-free herd that we maintained at the University ofWisconsin-Madison. Three cows (36-month-old Holstein, designated animals5695, 5970, and 6117) were bled by jugular venipuncture using bloodcollection bags (TERUFLEX, Somerset, N.J.) containing citrate phosphatedextrose adenine as an anticoagulant. Blood was transferred to 50-mlpolypropylene tubes and centrifuged at 1,400×g for 20 min at 25° C.Buffy coat containing white blood cells was isolated and mixed withphosphate-buffered saline (PBS) (Ca²⁺- and Mg²⁺-free) to a final volumeof 30 ml. The cell suspension was layered onto 58% isotonic PERCOLL(medium for density gradient centrifugation) (Sigma) at a 1:1 ratio andcentrifuged at 2,000×g for 30 min at 25° C. Peripheral blood mononuclearcells (PBMC) were collected from the PERCOLL-PBS interface and washedthree times with PBS to remove residual PERCOLL. To isolate bovinemonocyte-derived macrophages (MDMs), PBMC were resuspended in RPMI 1640(Sigma-Aldrich, St. Louis, Mo.) with 20% autologous serum andtransferred to TEFLON nonstick coating jars followed by incubation for 4days at 37° C. and 5% CO2. MDM cells were harvested, washed, and seededwith 2×10⁶ cells/well in 24-well plates with 5% autologous serum.Immediately before MDM cell infection, M. avium subsp. paratuberculosiscultures grown to mid-log phase (OD₆₀₀ of 0.4 to 0.6) were pelleted andresuspended in an appropriate volume of cell culture medium to achieve a50:1 multiplicity of infection (MOI). The cells were incubated at 37° C.with 5% CO2 for 3 h, and, subsequently, the monolayers were washed twotimes with warm PBS to remove extracellular bacteria, and RPMI 1640medium containing 5% autologous serum was added. The plates wereincubated at 37° C. for up to 8 days, and the culture medium wasreplaced with fresh medium at 4 days after infection. In another set ofexperiments, MDM cells were pretreated overnight (18 h) with 0.01 ug/mlrecombinant bovine IFN-γ (Kingfisher Biotech, St. Paul, Minn.) beforeinfection with M. avium subsp. paratuberculosis strains. Thisconcentration of IFN-γ was adequate to activate bovine monocytes (23).Bovine MDM cells were lysed at 1, 4, and 8 days postinfection for CFUplating with serial dilutions. Student's t test was used for statisticalanalysis, where P values of 0.05 were considered to be significant toevaluate differences in CFU counts.

J744A.1 cell culture infection. The mouse macrophage cells (J774A.1)were maintained in RPMI 1640 (Sigma-Aldrich, St. Louis, Mo.)supplemented with 5 to 10% heat-inactivated fetal bovine serum (FBS)(Sigma-Aldrich) in 75-cm² filter-cap tissue culture flasks (TechnoPlastic Products, Trasadingen, Switzerland) in a water-jacketedincubator (Thermo Scientific, Waltham, Mass.) at 37° C. with 5% CO₂.When confluent, cells were detached with a cell scraper and resuspended,and 10% of the cell suspension was replenished with fresh culture mediumevery 3 to 4 days.

Macrophages were seeded at 1.5×10⁷ cells per 15-mm cell culture dish(Techno Plastic Products) in 15 ml of culture medium as described above,30 to 36 h prior to infection, and were incubated at 37° C. with 5% CO₂.At least 5 dishes were seeded for each time point. For IFN-γ activationexperiments, old culture medium was discarded and 15 ml of fresh mediumwith 100 U/ml recombinant murine IFN-γ (Pepro Tech, Rocky Hill, N.J.)was added to each dish, 16 to 20 h prior to infection. An approximate109 CFU bacterial suspension from mid-log phase was mixed with 12 ml ofcell culture medium (RPMI 1640-10% FBS, mycobactin J-free) and added toeach decanted dish (MOI=50). The cells were incubated at 37° C. with 5%CO2 for 2 or 24 h before intracellular bacteria isolation. For the24-h-time-point experiments, extracellular bacteria were washed away at2 h postinfection with 15 ml of warm PBS at least five times or until novisible bacterial particles were observed under an inverted microscopeat ×400 magnification. The washed cells were replenished with 15 ml offresh cell culture medium and incubated until 24 h postinfection. Eachcondition was replicated at least twice until the quality of extractedRNA passed the criteria described below.

Immunofluorescent staining for LAMP-1 expression. Culture cells grown ona circular coverslip were fixed in 2.5% paraformaldehyde for 10 min andpermeated with cold methanol-acetone (1:1) at −20° C. for 5 min. Afewdrops of TB AuramineM(BD Diagnostics, Franklin Lakes, N.J.) were addedand incubated at room temperature for 10 min to stain mycobacteria. Thecoverslip was washed with 95% EtOH three times and rinsed with PBScontaining 0.2% saponin and 2% goat serum. Rat monoclonal antibody 1D4Bagainst mouse LAMP-1 purchased from the Developmental Studies HybridomaBank at the University of Iowa was diluted to 20 ug/ml inPBS-saponin-goat serum and incubated with the fixed cells at roomtemperature for 1 h. The cells were washed with PBS-saponin-goat serumthree times, each for 10 min. Goat antibody conjugated with ALEXA FLUOR633 fluorophore dye against rat IgG (Invitrogen, Carlsbad, Calif.) wasdiluted to 10 ug/ml in PBS-saponin-goat serum and incubated with thecells for 1 h in the dark at room temperature. The cells were thenwashed in the same way as described in the last step. Finally, thecoverslip was mounted on a microscope slide in Vectashield mountingmedium (Vector Laboratories, Burlingame, Calif.) and observed with aNikon C1 confocal microscope system.

Phagosome pH measurements. Phagosome pH measurement was slightlymodified from previous studies (26, 27) based on ratiometricmeasurements. J774A.1 cells were seeded at 2×10⁵ cells per well on a24-well cell culture plate (Techno Plastic Products) in 0.5 ml ofculture medium with or without 100 U/ml murine IFN-γ. A poly-L-lysine(Electron Microscopy Sciences, Hatfield, Pa.)-coated 12-mm circularcoverslip was placed in each well before seeding. After overnightincubation, culture medium was replaced with 0.3 ml of prewarmed freshmedium with 5 uM LYSOSENSOR radiometric probe Yellow/Blue DND-160(Invitrogen), and the cells were incubated for 5 min at 37° C. Togenerate an in situ pH gradient standard curve, each coverslip was thenincubated with morpholineethanesulfonic acid (MES) buffer (25 mM MES, 5mM NaCl, 115 mM KCl, and 1.2 mM MgSO4) of known pH (from 3.5 to 7.0 at a0.5 interval), in the presence of 10 uM nigericin and 10 uM monensin,for 2 min. The coverslip was immediately mounted on a glass slide andobserved under an OLYMPUS BX51 microscope with a reflected fluorescencesystem. Sixteen-bit grayscale images of two separate channels(excitation of 365/10 nm, emission of 460/50 nm, dichroic of 400 nm;excitation of 365/10 nm, emission of 540/20 nm, dichroic of 400 nm;Chroma, Bellows Falls, Ver.) from each field were taken.

The processing time from sample mounting to image acquisition wascontrolled so it took no longer than 10 min for each coverslip. Imageprocessing was done with ImageJ 1.44k (28). For each pH standard, atleast 20 individual regions of interest (ROIs) were randomly chosen, andmean intensities of each ROI from both channels were recorded. Ratios ofintensities of green (540 nm) to blue (460 nm) from the same pH standardwere then averaged, excluding values of =or >2 standard deviations (SD)from the mean. A standard curve of ratios was plotted against pH byapplying a Boltzmann equation, y=A2+(A1−A2)/{1+exp[(x−x0)/dx]}, where A1and A2 represent the limits of the fluorescent ratio at infinitely lowand high pHs, respectively, x0 is the pH midpoint at (A1+A2)/2, x is theobserved pH, and dx is the slope of the curve. When needed, cells wereinfected with M. avium subsp. paratuberculosis as described above,except the bacteria were prestained with 5 uM VYBRANT dye DiDcell-labeling solutions (Invitrogen) for 10 min (29). Intracellularbacteria could be observed with a third filter set (excitation of 535/50nm, emission of 675/20 nm, dichroic of 565 nm). ROIs were chosen wherethe bacteria were colocalized with LYSOSENSOR radiometric probe-stainedphagosomes. The average 540/460 ratio of ROIs was plugged into theequation to calculate phagosome pH.

Intracellular bacterial isolation and RNA extraction. Intracellularbacteria were isolated by a protocol described before (30) withmodifications. At 2 or 24 h post infection, infected cells were washedwith 15 ml ice-cold PBS at least five times or until no visiblebacterial particles were observed under an inverted microscope. Thewashed cells on each dish were then lysed with 10 ml cell lysis buffer(4 M guanidine thiocyanate, 0.5% sodium N-lauryl sarcosine, 25 mM sodiumcitrate, 0.5% TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), and0.1 M β-mercaptoethanol) and collected with a rubber cell scraper. Toreduce viscosity and help dissolve cell debris, cell lysates from alldishes were pooled and passed through a 23-gauge needle five times. Thelysate was then split into four 14-ml polypropylene centrifuge tubes(Falcon 352059; BD Biosciences, San Jose, Calif.) and centrifuged at3,200×g and 4° C. for 25 min. Each pellet was washed in 1 ml of TRIZOLregent (monophasic solution of phenol and guanidine isothiocyanate)(Invitrogen) twice and subjected to RNA extraction. Total RNA wasextracted by a protocol described before (12, 31). Briefly, bacterialpellets were resuspended in 2 ml of TRIZOL reagent (monophasic solutionof phenol and guanidine isothiocyanate) and split into two 2-mlscrew-cap tubes, each with 3.0 g of 0.1-mm zirconia/silica beads(BioSpect Products, Bartlesville, Okla.) and disrupted in aMini-BeadBeater-8 (BioSpect Products) at top speed for three pulses of60 s with 30-s intervals on ice. Following a 5-min incubation at roomtemperature, the supernatant was transferred to RNase-free tubes andcentrifuged at 12,000×g for 15 min. RNA was then isolated according tothe manufacturer's instruction. To remove genomic DNA contamination, RNAsamples were treated with 10 U of Turbo DNase (Ambion, Austin, Tex.) at37° C. for 30 min. An 1S900 241-bp PCR was performed to confirm that nogenomic DNA was detectable in the RNA samples (1). DNase treatments wererepeated if needed. Quality of the extracted RNA was examined with aNANODROP 1000 spectrophotometer (Thermo Scientific). The ratios ofA₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ must be higher than 1.8 and 1.5, respectively,before proceeding to cDNA synthesis for transcriptome studies.

Transcriptome studies. The NimbleGen (NimbleGen System Inc., Madison,Wis.) M. avium subsp. paratuberculosis microarray was designed from the4,350 open reading frame sequences in the genome of M. avium subsp.paratuberculosis (32). The whole genome was represented three times oneach chip. In addition, each gene was represented by 20 probes of 60-meroligonucleotides. As a result, each gene was represented by a total of60 probes. Double-stranded cDNA synthesis from isolated RNA samples,microarray hybridization, and data analysis were performed as previouslydescribed (12, 33). Significantly expressed genes were selected by±2-fold of change and P values of <0.05 by Student's t test. Theintensities were also exported to Gene Spring GX (Agilent Technologies,Santa Clara Calif.) for principal component analysis (PCA) on treatmentconditions, which is a method to reduce dimensionality in multiconditionmicroarray experiments and to find relevant patterns across conditions(34). Two biological replicates were included for each condition.

For RNA-seq studies, purified RNA samples were used for depletion ofrRNA sequences to enrich mRNA using the MICROBEXPRESS bacterial mRNAenrichment kit (Ambion, Austin, Tex.). Approximately, 10 ug of total RNAfor each sample was processed according to the manufacturer'sinstructions. For cDNA library preparation and sequencing, samplescontaining at least 1 ug of enriched mRNA were sent to the DNASequencing Facility at the University of Wisconsin-Madison BiotechnologyCenter. An Illumina HiSeq 2000 platform using one flow cell lane with100-cycle paired-end chemistry (Illumina, San Diego, Calif.) was used tosequence the cDNA library clusters. Raw RNA-seq data files in FASTQformat were assembled against the M. avium subsp. paratuberculosis (35)using the CLC Genomics Workbench 4.8 (CLC bio, Aarhus, Denmark). Geneexpression for each of the different sample conditions was calculatedusing “reads per kilobase million” (RPKM) expression values (36). Thefollowing formula was used to determine the RPKM values: RPKM=number ofreads/(kilobase length of gene×millions of mapped reads).

This RPKM metric enables comparisons between data sets with a varyingnumber of total reads. All reads mapping to annotated noncoding RNA(ncRNA) were removed from the data sets before determining RPKM values.Data sets were quantile normalized, and Kal's Z-test (37) was used forthe comparative gene expression analysis. Genes were consideredsignificantly, differentially expressed if they showed a ±2.5-foldchange with FDR P values of <0.05.

Quantitative real-time PCR. Quantitative real-time PCR (qRT-PCR) waspreviously described (12, 38) for confirmation of transcript levels. ASYBR green-based reagent with ROX (Bio-Rad, Hercules, Calif.) was usedwith 50 ng of double-stranded cDNA in each reaction. Double-strandedcDNA synthesis is described in the microarray sample preparationsession. No gDNA was detected from the RNA samples for cDNA synthesis.qRT-PCRs were performed with a 7300 real-time PCR system (AppliedBiosystems, Foster City, Calif.). The threshold cycle (CT) of each genewas normalized to the CT of the 16S rRNA gene from the same cDNA sample.The expression fold changes were calculated by comparing the normalizedCT of treated samples to the control sample as detailed before (39, 40).

Microarray data accession number. Data sets discussed in this reportwere deposited in NCBI's Gene Expression Omnibus (41) and are accessiblethrough GEO Series accession number GSE43645.

Results

Characterization of M. avium subsp. paratuberculosis-containingphagosomes. In our previous study, we defined the stressome of M. aviumsubsp. paratuberculosis under various in vitro conditions that mimickedthe hostile host microenvironments, including low pH and oxidativestress (12). In the present study, we further examined the bacterialresponses in the early stage of cell infection using a murine macrophageinfection model. Both naive and IFN-γ-activated cells were used in ourstudy. We monitored the expression of inducible nitric oxide synthase(iNOS), a marker for macrophage activation, with quantitative real-timePCR following IFN-γ treatment of J774A.1 cells. The transcriptionactivity of iNOS in IFN-γ-treated cells was over 1,000 times higher thannaive cells (data not shown). The temporal profile of iNOS expressionindicated that naive macrophages were activated by 2 h postinfection andthroughout the course of infection, with comparable mRNA levelsregardless of the viability of M. avium subsp. paratuberculosis bacilli.Activated macrophages had a similar profile, but the iNOS expressionlevels were between 1.6 to 2.6 times greater than those of infectednaive macrophages at each time point.

Additionally, we measured the phagosomal pH in both naive and activatedmacrophages using a dual-emission dye LYSOSENSOR radiometric probeDN-160 that emits fluorescent signals in a pH-dependent manner. Beforeinfection, naive and activated macrophages had similar lysosomal pHlevels ranging from 5.1 to 5.3. At 2 h postinfection, the pH inphagosomes containing heat-killed M. avium subsp. paratuberculosisdecreased below 4.0 regardless of cell activation status. However, thepH in phagosomes containing live M. avium subsp. paratuberculosisbacilli decreased just below the preinfection level (i.e., 4.8 to 5.0),suggesting the ability of live bacteria to prevent phagosomeacidification. Activated macrophages, but not naive ones, were able tocontinuously decrease the pH of phagosomes containing live bacilli up to4 h of postinfection. As the infection progressed (24 h), activatedmacrophages exhibited a better ability to maintain a lower pH level thannaive macrophages.

To examine the role of M. avium subsp. paratuberculosis on phagosomematuration, we examined the percentage of colocalization betweenintracellular M. avium subsp. Paratuberculosis and the lysosome markerLAMP-1. While heat-killed bacteria showed over 85% colocalization withLAMP-1, live M. avium subsp. paratuberculosis significantly reduced thepercentage of colocalization with LAMP-1 at 2 h postinfection(67.6%±5.5) in naive macrophages, suggesting live M. avium subsp.paratuberculosis is able to rapidly circumvent the hostile environmentand to delay phagosome maturation. The percentage of colocalization didnot significantly change in activated macrophages over the course of theexperiment (87.92%±5.32 and 83.7%±9.5 at 2 h and 24 h, respectively),suggesting that preactivated host cells have a better ability to controlinvading intracellular pathogens by means of phagosome maturation. Thereduced phagolysosome fusion of naive macrophages was restored to alevel (78.4%±6.8) similar to that of the preactivated phagosome(81.6%±8.8) at 24 h postinfection, as also evidenced by the increasediNOS expression level of the naive macrophage infection compared touninfected cells (data not shown). In general, both phagosomal pH andLAMP-1 colocalization indicated the ability of live, virulent M. aviumsubsp. paratuberculosis to prevent phagosome acidification and to delaylysosomal fusion by 2 h postinfection.

Transcriptional profiling of M. avium subsp. paratuberculosis isolatedfrom infected macrophages. To profile changes in the levels of M. aviumsubsp. paratuberculosis transcripts within macrophages, we isolatedintracellular bacilli at 2 and 24 h postinfection, with or without IFN-γactivation. Because the bacteria must stay in the cell culture medium(RPMI 1640-10% FBS, mycobactin J-free) before they can infect hostmacrophages, we compared the transcriptomes of intracellular bacteria tothose incubated in vitro in cell culture medium for 2 h. Under allconditions tested, the correlation coefficients (r) between biologicalreplicates ranged between 0.92 and 0.99. To examine the statisticaldistance between each biological replicate and among treatments, athree-dimensional principal component analysis plot was generated,indicating high correlations between biological replicates. Clusteranalysis identified groups of genes active only during macrophageinfection. Compared to the RPMI-incubated control sample, expressionlevels of 136 and 333 M. avium subsp. paratuberculosis genes weresignificantly changed in naive macrophages at 2 and 24 h postinfection,respectively. On the other hand, in IFN-γ-activated macrophages, thenumbers of genes with significantly changed expression levels were 284and 328, respectively. Among those genes, 47 were common in all of the 4examined macrophage conditions, representing a core set of genesresponsible for interacting with the macrophage microenvironment.

In general, M. avium subsp. paratuberculosis transcriptomes in infectedmacrophages preactivated with IFN-γ were more similar to those observedunder in vitro stressors reported earlier (12). Also, IFN-γ activationof macrophages resulted in significant induction of a group of genes(n=48), mostly those involved in energy production and conversion (e.g.,icl, fdxA, sdhABCD, and ndh) or nutrient transport and metabolism (e.g.,fad genes, dapA_1, and cysH_2), at 2 h postinfection in IFN-γ-activatedmacrophages compared to naive macrophages.

At 24 h postinfection, we started to see a significant change in M.avium subsp. paratuberculosis transcriptomes indicative of change oftheir microenvironment, especially in activated macrophages. Forexample, the mbt operon (mbtA to mbtE and MAP2172c) involved in ironmetabolism was significantly upregulated at 24 h postinfection inIFN-γ-treated macrophages compared to the RPMI-incubated controlsamples. A similar observation was reported for 120 h postinfection ofbovine monocyte-derived macrophages (MDMs) (21). MAP2172c was shown tobe repressed in M. avium subsp. paratuberculosis cultures grown inmycobactin J-depleted medium over time, where expression levels of othermbt genes remained constant (42). This paradigm could suggest that anintracellular environment is not iron exochelin-free, or there are otherintracellular factors that stimulate alternative iron metabolicpathways, at least in the examined times. On the other hand, a geneinvolved in iron storage, bfrA (43), was significantly downregulated atthe same time point, while the mbt operon was activated, suggesting thelack of access to iron inside the mycobacterial phagosome.Interestingly, the ex-pression of the iron-dependent regulator ideR (43)remained unchanged during the examined time course regardless ofmacrophage activation status, suggesting a lesser role of ideR in earlystages of macrophage infection. Overall, a significant time-dependentshift in M. avium subsp. paratuberculosis transcriptomes was evidentfrom examining M. avium subsp. paratuberculosis collected at 2 and 24 hafter macrophage infection.

Microenvironment of M. avium subsp. paratuberculosis. One of our goalsis to gain more insights into the intracellular environment of M. aviumsubsp. paratuberculosis using transcriptome analysis. Schnappinger etal. reported that M. tuberculosis upregulates (3-oxidation genes by 4 hpostinfection in murine bone marrow macrophages, suggesting a transitionof carbon source from carbohydrates to fatty acids (44). Similarly, ourstudy indicated activation of M. avium subsp. paratuberculosisorthologues (fadA6_3, fadB_1, fadD9, fadE17, fadE21, fadE3_1, and fadE5)in the β-oxidation pathway starting from as early as 2 h postinfection,suggesting the transition of carbon source utilization is a commonbacterial strategy between M. tuberculosis and M. avium subsp.paratuberculosis. The id gene, previously known as aceA, was also amongthe highly upregulated genes involved in carbon metabolism. The geneproduct, an isocitrate lyase, bridges the β-oxidation pathway toglyoxylate cycle, an anabolic pathway with a net product of glucose. Thecontribution of icl to M. avium subsp. paratuberculosis survival inmacrophages remains to be analyzed.

Once entering host cell compartments, intracellular bacteria encounterhost defense mechanisms such as reactive oxygen intermediates (ROIs),reactive nitrogen intermediates (RNIs), digestive enzymes, and, mostimportantly, lower pH. In addition, the phagosome is also anutrient-depleted environment. Accordingly, we examined genes that areassociated with stress response and intracellular bacterial survival.Several known oxidative stress-induced genes, such as oxyR, trxB, trxC,tpx, ahpC, and ahpD, were significantly upregulated in all intracellularconditions. OxyR is a redox sensor protein that, when oxidized,positively regulates a group of genes, including ahpC, katG, gorA, andfurA (45). Among those genes, the ahpCD operon was highly upregulated(6.2- to 11.0-fold) in samples taken from naive or activatedmacrophages. In addition, TrxB, TrxC, and Tpx, proteins involved inreduction of thio-disulfide and resistance of hydroperoxide processes(46), were upregulated, suggesting active machinery for counteractingoxidative stress within host cells. However, other known oxidativeresponsive genes, such as katG, gorA, furA, sodA, and sodC, were notactivated in these samples, possibly because those genes are indirectlyregulated by oxyR or also under the control of other stress-responseregulators. Overall, M. avium subsp. paratuberculosis deployed specificgene products to defend against the hostile microenvironment during thisearly stage of infection.

Changes in global gene regulators. Sigma factors play a central role inbacterial gene regulation (47) and pathogenesis as reviewed in M.tuberculosis (48) and other pathogens (49). Nineteen sigma factors wereidentified in M. avium subsp. paratuberculosis by sequence analysis(24), 13 of which are homologous to M. tuberculosis sigma factors. ThesigA gene, though considered a constitutively expressed sigma factorgene in M. tuberculosis (50), was found downregulated to nearly 2-foldat 24 h in activated macrophages. The sigB gene, which is a dispensablesigma factor and partially responsive to some oxidative and heat shockstresses in M. tuberculosis (51), showed a slight increase (˜1.6-fold)at 24 h postinfection under both activated and naive states ofmacrophages. Genes of sigC, sigG, sigJ, sigM, and other extracytoplasmicsigma factors (ECF-2 through ECF-6) remained constantly expressed in allexamined conditions. However, as shown in FIG. 1, sigH was the mostinduced among other sigma factors of M. avium subsp. paratuberculosis,and the activation seems to be augmented by macrophage activation overtime. sigH transcripts were upregulated under in vitro heat shock andoxidative stress treatments in M. avium subsp. paratuberculosis (12).Also, sigL was upregulated at the 2-h time point but downregulated by 24h postinfection, suggesting a potential role for sigL in the very earlystage of infection. On the contrary, sigE expression was significantlyupregulated after 2 h postinfection and remained high at 24 hpostinfection, suggesting a prolonged role during M. avium subsp.paratuberculosis persistence. Overall, a few sigma factors showed adynamic and active gene regulation transition during the first 24 hpostinfection within macrophages. It is possible that other regulatorscould play a similar role during later times of infection or indifferent host cells.

Role of sigH in M. avium subsp. paratuberculosis to variable stressconditions. The sigH gene in M. tuberculosis has been shown to beupregulated upon heat shock, upon diamide treatment (25), and duringsurvival in human macrophages (52), suggesting its importance inresponding to extracellular stimuli and intracellular survival. To testthe hypothesis that sigH could play an important role in M. avium subsp.paratuberculosis stress responses, we employed a specializedtransduction protocol (53) to generate a sigH isogenic knockout mutantin the M. avium subsp. paratuberculosis K10 genetic background. BecausesigH and its anti-sigma factor (MAP3323c) are likely encoded in anoperon (54), the ΔsigH mutant was examined for possible polar effects onthe downstream gene, MAP3323c. Using reverse transcriptase PCR, thepresence of the MAP3323c transcript was confirmed in the ΔsigH mutantstrain.

After construction, the resistance of the ΔsigH mutant was evaluatedagainst various stressors. Analysis of the disc diffusion assay revealedthat the ΔsigH mutant does not tolerate thiol-specific oxidationcompared to the wild-type strain, as evidenced by the observed halozones on plates. However, no other differential resistance was observedwhen a cell wall stressor (sodium dodecyl sulfate) or ethambutol discswere used (data not shown). To measure viability of the ΔsigH mutantafter sustained exposure to diamide or heat stress, we cultured bothwild-type and the mutant strains for an extended time period in thepresence of diamide or a 45° C. water bath. In both stress conditions,there was significant reduction at each time point in the viability ofthe ΔsigH mutant compared to that of the wild-type strain. At day 7, theviability of the ΔsigH mutant was reduced by almost 2-log orders andmore than 1-log order in CFU counts relative to the wild-type strain fordiamide and heat stress, respectively. Unfortunately, replicativeplasmid complementation of the ΔsigH mutant with a wild-type sigH underthe control of the hsp65 promoter did not restore the diamide resistancephenotype (data not shown), most likely due to inefficientcomplementation in mycobacterial strains (25, 55, 56).

Intracellular survival of the ΔsigH mutant in bovine MDM cells.Intracellular growth kinetics of M. avium subsp. paratuberculosisstrains were analyzed using bovine MDM cells. MDM cells were infectedwith the ΔsigH mutant and its parental strain for a prolonged time up to8 days after infection. The MDM monolayer in the culture wells waschecked at a regular interval for cell confluence under an invertedlight microscope. We first determined intracellular viability of boththe ΔsigH mutant and wild-type strains within the naive MDM cells. Thenumbers of wild-type strain of M. avium subsp. paratuberculosis bacilliincreased 2-fold, whereas the growth of the ΔsigH mutant was notsupported within naive MDM cells as determined by CFU plating at 8 daysafter infection (FIG. 2A). Next, we examined whether the ΔsigH mutantwould be able to survive inside activated MDM cells pretreated withrecombinant IFN-γ. At 8 days post infection, there was more than a2-fold increase in the number of wild-type M. avium subsp.paratuberculosis bacilli, whereas in the IFN-γ pretreated MDM cells,viability of the ΔsigH mutant was significantly reduced almost by 50%(FIG. 2B). These observations suggested an important role for sigH indefending M. avium subsp. paratuberculosis against IFN-γ activation.

Virulence analysis of the M. avium subsp. paratuberculosis ΔsigH mutant.To assess the role of SigH in M. avium subsp. paratuberculosisvirulence, we investigated the persistence of the M. avium subsp.paratuberculosis ΔsigH mutant using the mouse model of paratuberculosis.The initial growth kinetics of the wildtype and ΔsigH mutant strainswere similar, with an equal burden of bacteria in both intestine andspleen up to 6 wpi (FIG. 3). However, the colonization levels of theΔsigH mutant compared to its parental strain were significantly reducedin spleen and intestine at 12 wpi, suggesting a role for sigH in thelong-term survival of M. avium subsp. paratuberculosis in mice.Interestingly, when M. tuberculosis ΔsigH was used to challenge mice, nodifferences in bacterial load were observed in mouse organs compared tothat of the wild-type strain (57).

Evaluation of the hematoxylin and eosin-stained spleen, liver, andintestine organs at 3, 6, and 12 wpi showed moderately similar tissuepathology when infected with the ΔsigH mutant or wildtype M. aviumsubsp. paratuberculosis. Granulomatous inflammation was evident in theliver tissues by 12 wpi, with no visual differences in mycobacterialcolonization among the mouse groups infected with wild-type or mutantstrains. However, mouse spleen tissues infected with the wild-typestrain displayed higher follicular atrophy than the spleen tissuesinfected with the ΔsigH mutant. Consistent with the bacterialcolonization data, Ziehl-Neelsen staining showed higher numbers ofacid-fast bacilli in the mouse spleen infected with the wild-type strainthan the ΔsigH mutant at 12 wpi (FIG. 3C). Taken together, our dataindicated that the ΔsigH mutant was attenuated in the murine model ofparatuberculosis compared to the wild-type M. avium subsp.paratuberculosis strain.

Transcriptional regulation of sigH in M. avium subsp. paratuberculosis.Our stress experiments showed that the ΔsigH mutant was hypersensitiveto elevated temperature and diamide exposure, each resulting in impairedgrowth. On the basis of these findings, we hypothesized that sigH mayplay an important role in directing transcriptional control underunfavorable environmental conditions. To identify gene regulatorynetworks under the control of sigH, both wild-type M. avium subsp.paratuberculosis and ΔsigH mutant transcriptomes were profiled beforeand after diamide exposure using the next-generation RNA sequencing(RNA-seq). When the wild-type strain was compared to the ΔsigH mutant,approximately 15% of the M. avium subsp. paratuberculosis genes (˜307induced and ˜344 repressed) were found to be differentially regulated at3 h postexposure to diamide stress. This large number of geneperturbation was likely orchestrated by additional sigma factors (e.g.,sigB, sigD, sigE) along with various transcriptional regulators thatwere differentially expressed in examined samples. Genes were groupedinto different functional categories, and a large number of genes (e.g.,hsp, clpB) belonging to the chaperonin functional category weresignificantly upregulated. Many induced genes were involved indetoxification and maintaining cellular redox homeostasis (e.g., trxB2,adhE) during oxidative stress as detailed before (46, 58). Manytranscriptional factors and two-component systems were found to beupregulated (e.g., sigB, sigE, whiB4, MAPK_0206, mtrA) under the controlof SigH. The expression of mycobacterial sigB and sigE was known to belinked with the presence of the sigH gene (59). WhiB-like proteins areredox-responsive DNA binding factors and could play a protective roleagainst oxidative stress (60). In mycobacteria, the role of theMtrB-MtrA two-component system is not entirely understood, but it wasfound to be essential for bacterial viability, particularly involved inthe regulation of cell wall permeability (61, 62).

A number of induced genes in the SigH regulon were related to virulence,and many of them were included in the mce gene family (e.g., mceA1,mceC, mceD). Several mce genes were shown to be upregulated duringphagocytosis and oxidative stress exposure (52, 63), indicating thatthey are active during infection. Other key functional gene categorieswere associated with central intermediary/sulfate metabolism (e.g.,rm1B, rm1C, cysQ_2, cysD), energy metabolism (e.g., rpi, tpi, nuoA), andcell processes/transport (e.g., fdxC_2, MAPK_4062) or were cell enveloperelated (e.g., mmpL4_1, mmpS3). Our results indicate that many of thesegenes were induced under intraphagosomal stresses inside macrophages.Genes belonging to functional categories, including lipid metabolism(e.g., fadE14, fadD33_2, MAPK_2213), polyketides (e.g., pks2, papA3_2),and biosynthesis of amino acids (e.g., leuC, metA, trpE2), were amongthe down regulated genes in M. avium subsp. paratuberculosis relative tothe ΔsigH mutant following diamide stress. We also examined thedifferential expression profile of M. avium subsp. paratuberculosis inthe absence of sigH during standard physiological growth conditions(mid-log phase). In this case, gene categories belong to lipidmetabolism (e.g., fadD29), cell processes (e.g., kdpA, pstS),transcriptional regulation (e.g., MAPK_0788), and electron transport(e.g., fdxC_2). Additionally, we found that a large number of genesbelong to the hypothetical functional category (˜30%). To verify thetranscriptome results, a few upregulated genes were randomly selected,and qRTPCR was employed using the SYBR green method. The transcriptlevels of these genes analyzed by RNA-seq and qRT-PCR were in goodagreement and corroborated with the transcriptome data.

Identification of sigH-regulated promoters in M. avium subsp.paratuberculosis. For identification of promoters that were likely to bedirectly controlled by sigH, we analyzed a list of candidate transcriptswith higher expression ratios (wild type/ΔsigH mutant). Since SigH of M.avium subsp. paratuberculosis is a very close homologue of M.tuberculosis SigH (59), we searched for the presence of the consensussequence of M. tuberculosis SigH-dependent nondegenerate promoter motifsGGAA-N18-20-GTT in the 250-bp region upstream of start codons of M.avium subsp. paratuberculosis genes using the Genolist webserver(genodb.pasteur.fr/cgi-bin/WebObjects/GenoList). A total of 30 geneswere found to be directly upregulated by SigH with GGAA and GTT coremotifs at the −35 and −10 regions, respectively, in their promoterregions. Many of these targets of SigH in M. avium subsp.paratuberculosis were also found to be controlled in Corynebacteriumglutamicum (64), Streptomyces coelicolor (65), and M. tuberculosis (59),suggesting a conserved regulon directly controlled by SigH across thehigh-percentage GC Gram-positive actinobacteria.

Discussion

The intracellular pathogen M. avium subsp. paratuberculosis is known toinfect and persist within host macrophages with unclear mechanisms. Toexamine how M. avium subsp. paratuberculosis responds to intracellularenvironments, especially during the early stages of infection, we used amacrophage cell line coupled with DNA microarrays to profilemacrophage-induced changes in M. avium subsp. paratuberculosistranscriptome. A clear advantage of this infection model is theflexibility to control the activation status of the host cells inaddition to the availability of reagents and protocols for manipulation.By comparing the results of phagosome pH and phagosome colocalizationmarkers, we found significant differences in intracellular environmentsof naive versus active macrophages consistent with earlier studies (44,66). Activated macrophages, at the time of infection, showed much higheriNOS gene expression than naive macrophages. At 2 or 24 h postinfection,they showed higher phagosome colocalization with ingested M. aviumsubsp. paratuberculosis particles, which clearly exhibited a better celldefense mechanism than naive macrophages. However, during the course ofinfection up to 24 h, overall survival of intracellular M. avium subsp.paratuberculosis did not differ in either naive or activatedmacrophages. This phenotype could change later during persistentinfection which we did not address in this study. Most of thedifferentially expressed genes between these states are core stressresponsive genes involved in energy production, indicating M. aviumsubsp. paratuberculosis initiates stress responses to a higher levelmore rapidly in activated intracellular environments. On the host side,once activated, the host cells maintained their activation statusthroughout the course of infection. This suggests that virulent M. aviumsubsp. paratuberculosis has the ability to prevent phagosome maturationand subsequently circumvent detrimental low pH and oxidative stressesduring the very early stages of infection, possibly without interferingwith the host early signal transduction pathways responsible formacrophage activation.

When M. avium subsp. paratuberculosis transcriptomes in macrophages werecompared to our previous study of the in vitro stressome (12), therewere more common genes with the 24-h than 2-h-postinfection samples,indicating that during the early stage of infection, M. avium subsp.paratuberculosis is adjusting to more acidic and oxidizing environments.We also observed the metabolic shift of M. avium subsp. paratuberculosisto utilize fatty acids as the major carbon source, which has alreadybeen observed in M. tuberculosis (44) and M. avium subsp.paratuberculosis (21). The shift of metabolic activity at earlyinfection may be a common theme employed by mycobacterial pathogensunder nutrient-depleted conditions. By 24 h postinfection, securing ironfor M. avium subsp. paratuberculosis became a significant quest,especially in activated macrophages, as suggested by the activation ofthe mbt operon. It is well established that the phagosome is aniron-depleted compartment (44) and intracellular pathogens have evolvedways to scavenge iron within mammalian cells. However, iron acquisitionmechanisms of M. avium subsp. paratuberculosis remain unknown given thatM. avium subsp. paratuberculosis possesses a truncated mtbA gene andthus is unable to produce mycobactin (32).

Because of the important role played by global gene regulators inbacterial pathogenesis, we focused our analysis on the expressionprofile of the 19 sigma factors encoded in the M. avium subsp.paratuberculosis genome (32). Accordingly, in the experiments reportedhere, we were able to capture active gene regulation of a set of sigmafactors (e.g., sigH, ECF-1) during early macrophage infection with M.avium subsp. paratuberculosis. Consistent with the M. tuberculosisinfection studies, we found an immediate upregulation of sigH withinmacrophages. This trend continued through 24 h postinfection andindicated a crucial role of sigH to regulate stress-responsive genes,especially those activated during exposure to thiol oxidation, asindicated by the disc diffusion assay. Moreover, we have demonstratedthat the ΔsigH mutant is very sensitive to sustained exposure to diamideor heat stress compared to the wild-type strain. The sigE gene, anotherstress-induced sigma factor, did not show higher expression levels until24 h postinfection in either naive or activated macrophages. Thisdelayed response of sigE as well as modest induction of sigB mayindicate an indirect regulation by other immediate stress-responsivegenes and support the important role played by sigma factors inmycobacterial pathogenesis (25, 59). The other sigma factor that wasupregulated throughout the examined time course was ECF-1. This sigmafactor may also play an important role immediately upon infection, whichwas not reported before.

We have also profiled the regulatory network under the control of sigHby studying the relative abundance of gene transcripts using RNA-seq. Wefound that a large number of M. avium subsp. paratuberculosis genes weredirectly or indirectly regulated by sigH after exposure to diamidestress. In fact, analysis of the upstream sequence of the upregulatedgenes revealed a set of genes that could be directly controlled by SigH.Among them, many genes are involved in the functional category of heatshock and protein processing. Heat shock proteins (e.g., Hsp, DnaJ2,ClpB) are found widely on prokaryotic cells and act as molecularchaperones helping to configure proteins correctly upon encountering anunfavorable milieu (67, 68). Such environments, i.e., oxidative stress,could result in nonrepairable protein structures which may necessitatefull degradation by the C1pC protease (63). Oxidative stress scavengersinduced following diamide stress include TrxB2, TrxC, and AdhE (46, 58).All of these genes likely play important roles in redox homeostasisunder thiol oxidation and are found in high levels inside themacrophages (44, 63). Interestingly, the effect of diamide stress in M.tuberculosis also resulted in a transcriptional profile similar to thatof M. avium subsp. paratuberculosis (63), indicating the pivotal role ofsigH across mycobacterial species.

Consistent with the estimated large regulon of SigH, a significantdifference in survival rates between the ΔsigH and wild-type strains wasobserved inside activated bovine macrophages. Intracellular growth ofthe wild-type M. avium subsp. paratuberculosis strain was not inhibitedregardless of the activation state up to 8 days postinfection. Thisobservation was in corroboration with the earlier findings which showedthat activated bovine monocytes were inadequate to inhibit intracellulargrowth of M. avium subsp. paratuberculosis up to 9 days after infectionas determined by the CFU method (69). In contrast, viability of theΔsigH mutant was significantly impaired, indicating an importantfunction of sigH for the intracellular growth of M. avium subsp.paratuberculosis, possibly by blocking IFN-γ activity as suggestedearlier (23). In recent studies, clues have been obtained on macrophageinteraction with M. avium subsp. paratuberculosis that indicate thecapacity of this pathogen to subvert host immune responses by blockingthe ability of mononuclear phagocyte maturation (23, 70, 71). Althoughit is tempting to speculate that the ΔsigH mutant failed to interferewith macrophage maturation, especially when preactivated with IFN-γ,more experiments are needed to fully understand the mechanisms that sigHcould play during macrophage infection. The survival profile of M. aviumsubsp. paratuberculosis constructs in MDM was further supported by theinability of the ΔsigH mutant to survive in mice. Both bacteriologicaland histological analyses displayed impaired organ colonization of theΔsigH mutant with a low inflammatory response. However, we did not findany comparative differences in liver organs infected with the wild-typeand ΔsigH mutant strains. A recent study showed that the M. tuberculosisΔsigH mutant was completely attenuated in nonhuman primates (72), abetter experimental model than mice for studying human tuberculosis(57). It will be interesting and important to examine the survival ofthe M. avium subsp. paratuberculosis ΔsigH mutant in a ruminant model ofparatuberculosis (e.g., goat).

In conclusion, our analyses indicated significant changes inmycobacterial gene expression during macrophage survival, most likelyunder the control of sigH and other sigma factors. The activation statusof macrophages also directs the mycobacterial response to a specificstress-responsive profile. We demonstrated that sigH offers a massivetemporal response on the M. avium subsp. paratuberculosis transcriptometo cope with the adverse effects of oxidative stress. Our data indicatethat sigH could play a critical role during infection, and activation ofits regulon is required for replication and full virulence of M. aviumsubsp. paratuberculosis. Further interrogation of these sigma factorsand their regulatory networks should ultimately furnish a greaterunderstanding of M. avium subsp. paratuberculosis pathogenesis and helpdesign a better approach for controlling Johne's disease.

Example 2

References cited in this Example are listed in the section of Referencesas “References cited in Example 2.”

Materials and Methods

Bacterial strains. M. avium subsp. paratuberculosis K10 and M. smegmatismc²155 strains were grown in Middlebrook 7H9 broth and on Middlebrook7H10 plates as previously described (2,19). For cloning, Escherichiacoli DH5a and HB101 were used as host cells. To generate an M. aviumsubsp. paratuberculosis mutant, a specialized transduction protocol wasadopted to delete the sigL/MAP4201 gene using the M. avium subsp.paratuberculosis K10 strain (19,25). Primers were designed to amplify˜750 bp PCR fragments flanking each end of the sigL coding region andcloned into pYUB854 (19). The resulting vector was used to generate sigLdeleted mutant, M. avium subsp. paratuberculosis ΔsigL, according to themethods described elsewhere (26). Genotype of the M. avium subsp.paratuberculosis ΔsigL was confirmed by PCR and sequence analysis (19).To complement the M. avium subsp. paratuberculosis ΔsigL, a ˜4-kbfragment, encompassing sigL with its 5′ regulatory region and the distalgenes (MAP4202-MAP4205), was amplified by PCR and cloned into pMV306(24). The M. avium subsp. paratuberculosis ΔsigL strain was transformedwith this recombinant construct and genotype of the complemented strain(M. avium subsp. paratuberculosis ΔsigL::sigL) was identified by PCRanalysis. A similar approach was applied to complement M. tuberculosismutant strain lacking such alternative sigma factor (27).

Stress phenotype of M. avium subsp. paratuberculosis. M. avium subsp.paratuberculosis cultures were grown to log phase (OD₆₀₀=0.5-1.0) and200 μl spread on 7H10 plates. For disk diffusion assay (DDA), paperdiscs (6-mm, Whatman, Piscataway, N.J.) containing 20 μl of 0.5, 1, or1.5M diamide (oxidative stressor) and 1, 2, or 3% sodium dodecyl sulfate(SDS; cell wall stressor) were placed on each of the spread plate.Plates were incubated at 37° C. until a thick confluent lawn developed.To determine sustained effect of stressor on the viability of bacilli,after washing with PBS, M. avium subsp. paratuberculosis cultures wereexposed to the acidified 7H9 broth (pH 5.5 obtained by adding HCl)containing 0.3% bovine bile (cell wall stressor) and aliquots werecollected at 0, 4, 15, 24, 48, and 72 h to monitor their viability bycolony forming units (CFU) (19).

Cell culture and infection. The mouse macrophage cells (J774A.1) wereregularly maintained as described elsewhere (19). To activatemacrophages, cells were pretreated overnight (18 h) with 100 U/mlrecombinant murine IFN-γ (Pepro Tech, Rocky Hill, N.J.) before infectionwith M. avium subsp. paratuberculosis strains (19). For cell infectionstudies, wild-type and mutant strains were added to macrophagemonolayers (multiplicity of infection [MOI], 20:1). Following incubationat 37° C. in 5% CO₂ for 3 h, macrophage monolayers were washed twicewith warm PBS to remove extracellular bacteria and RPMI-1640 mediumcontaining 5% fetal bovine serum was added. Cells were lysed at 1, and 8days post-infection for bacterial CFU counts. To examine M. avium subsp.paratuberculosis ΔsigL survival in bovine monocyte-derived macrophages(MDM), MDM cells were isolated from peripheral blood of three cows andcell infection studies were performed as described in detail elsewhere(19).

Mouse infections. All animal experiments used in this study wereperformed according to the protocols approved by the InstitutionalAnimal Care and Use Committee, UW-Madison. For the virulence study, twogroups (N=15 per group) of female BALB/c mice (Harlan Laboratories,Indianapolis, USA) were challenged intraperitoneally (i.p) with thewild-type and mutant strains. Infection inocula (˜2×10⁸ CFU/mouse) ofthe two strains were similar as determined by plate count on the day ofinfection. Mouse groups (N=5) were sacrificed at 3, 6 and 12 weekspost-infection (WPI), and organ samples were collected for bacterial CFUenumeration and histopathological examinations as described before (19).For the immunization studies, female C57BL/6 mice (Taconic, Hudson,N.Y.) were used. Mock group (N=12) was immunized with PBS buffer whileM. avium subsp. paratuberculosis ΔsigL mice group (N=14) received ˜2×10⁶CFU in 0.2 ml PBS subcutaneously (s.c.) into the neck scruff twice, 2weeks apart. Four weeks following booster dose, mice were challengedi.p. with ˜7×10⁸ CFU wild-type M. avium subsp. paratuberculosis strainas determined by plate count on the day of infection. Mouse groups(N=4-6) were sacrificed at 6 weeks post-immunization and 6 and 12 weekspost-challenge (WPC), and organ samples were collected for bacterial CFUcounts, histopathological examinations, and immune responses.

Evaluation of immune responses. Mouse spleens were collected asepticallyand homogenized by gentle mechanical disruption. Following spleen cellisolation, splenocytes were cultured in duplicate in round bottom96-well tissue culture plates with 1×10⁶ cells/well (28). Cells werere-stimulated in vitro with 10 μg/ml Johnin purified protein derivative(PPD) (NVSL, Ames, Iowa) for 48 and 72 h. Cell supernatants werecollected and analyzed for cytokines by ELISA kit according to themanufacturer's instructions (BioLegend, San Diego, Calif.). To determinehumoral immune response, sera were prepared from mouse blood and M.avium subsp. paratuberculosis specific antibody (anti-PPDj antibodies)was detected by ELISA using Horseradish peroxidase conjugated rabbitanti-mouse antibody (Pierce, Rockford, Ill.) (29).

Statistical analysis. Student's t test was performed to comparedifferences in mouse immune responses and bacterial CFU counts from invitro stress treatments. Mann-Whitney U test was used to comparebacterial loads in mouse organs. A probability value of <0.05 wasconsidered significant.

Results

Effect of sigL mutation on viability of M. avium subsp. paratuberculosisunder stress. Recent analysis of M. avium subsp. paratuberculosistranscriptome during macrophage infection suggested that sigL could bean important factor for M. avium subsp. paratuberculosis survival insidehost macrophages (19). To test this hypothesis, we generated a sigLdeletion mutant, M. avium subsp. paratuberculosis ΔsigL (FIG. 4A-B) andexamined survival of this mutant under different stress conditions.Because sigL and its anti-sigma factor (MAP4202) are likely encoded inan operon (24), we examined M. avium subsp. paratuberculosis ΔsigLstrain for possible polarity on the downstream gene, MAP4202. Byemploying reverse-transcriptase PCR analysis, presence of the MAP4202transcript was confirmed in the ΔsigL mutant (FIG. 4C).

To examine a potential role for sigL in M. avium subsp. paratuberculosisresponse to unfavorable stress conditions, we analyzed the survival ofM. avium subsp. paratuberculosis cultures under both oxidative (diamide)and cell wall stresses (SDS and bovine bile). Growth inhibition zones indisk diffusion assays indicated that M. avium subsp. paratuberculosisΔsigL was susceptible to diamide oxidation. Such phenotypic differencesalso indicated the inability of M. avium subsp. paratuberculosis ΔsigLto survive under SDS stress when compared to the wild-type andcomplemented strains. Bile tolerance was also evaluated by culturing ofthe M. avium subsp. paratuberculosis strains in the presence of 0.3%bovine bile (oxgall). This concentration of bile is likely encounteredby the bacteria within the intestinal content following oral infection(30). In addition, because of the ability of M. avium subsp.paratuberculosis to resist killing by acidic conditions (31), we madeculture broths slightly acidic (pH 5.5) to partially mimic thephysiological condition that M. avium subsp. paratuberculosis wouldencounter following infection in the gastrointestinal tract (e.g.abomasum of a cow and stomach of a human). Survival levels showed asignificant drop in the viability of the M. avium subsp.paratuberculosis ΔsigL at 4 h post-exposure to bovine bile compared tothe wild-type and complemented strains. This difference in bacterialsurvival for M. avium subsp. paratuberculosis ΔsigL was increased bymore than 1.0 log at 24 h and the viability of the mutant continued todecline at later times suggesting that sigL is important in renderingresistance when bacteria experience initial bactericidal barriers in thehost. Because complementation of the M. avium subsp. paratuberculosisΔsigL restored wild-type phenotype under these stress conditions, thecomplemented strain was not included in further experiments.

Intracellular survival within macrophages. Because sigL was up-regulatedinside activated murine macrophages (19), we examined intracellularsurvival of M. avium subsp. paratuberculosis ΔsigL in the IFN-γpretreated murine macrophages. Our analysis showed an increase in thenumber of wild-type M. avium subsp. paratuberculosis at 8 days relativeto the numbers obtained at day 1 post-infection whereas viability of theM. avium subsp. paratuberculosis ΔsigL was significantly reduced at thistime. To use a more relevant model for M. avium subsp. paratuberculosisinfection, we evaluated the persistence of the M. avium subsp.paratuberculosis ΔsigL both in resting and IFN-γ-activated bovinemonocyte-derived macrophages (MDM cells), the natural host cell for M.avium subsp. paratuberculosis. At 8 days post-infection, the number ofwild-type bacilli increased over twofold compared to the numbersobtained at day 1 in the resting MDM cells. Specifically, naive andIFN-γ pretreated MDM cells were infected with M. avium subsp.paratuberculosis ΔsigL and wild-type M. avium subsp. paratuberculosisstrains. Cells were lysed at 1, and 8 days post-infection and numbers ofviable bacilli were determined by serial dilutions for CFU plating. Thesurvival level at 8 days was relative to the viable counts of bacterialstrains at day 1. Survival data represent the average of macrophageinfections collected from three different donor animals withsignificance levels in Student's t test (*p<0.05).

In contrast, viability of the M. avium subsp. paratuberculosis ΔsigL wassignificantly reduced almost by half indicating a potential function forsigL in defending M. avium subsp. paratuberculosis against intracellularstress. A similar survival trend for the M. avium subsp.paratuberculosis ΔsigL was seen inside IFN-γ pretreated MDM cellswhereas this activation status did not result in more inhibitory effecton the survival of wild-type bacilli. Collectively, survival assaysindicated that deletion of sigL affected M. avium subsp.paratuberculosis viability following exposure to stress conditionssuggesting a significant function for sigL in defending M. avium subsp.paratuberculosis against intracellular insults.

Virulence analysis of M. avium subsp. paratuberculosis ΔsigL strain. Toevaluate the contribution of SigL to M. avium subsp. paratuberculosisvirulence, we examined persistence of the M. avium subsp.paratuberculosis ΔsigL using the murine model of paratuberculosis. Thesurvival pattern indicated significant attenuation for M. avium subsp.paratuberculosis ΔsigL as early as 3 WPI in all of the examined organs.In the spleen, viability of M. avium subsp. paratuberculosis ΔsigL wasreduced by more than 1-log and 2-log orders relative to the wild-typestrain at 6 and 12 WPI, respectively. Similarly, colonization levels ofthe mutant strain in the liver were significantly lower compared to theparental strain at all examined time points. Interestingly, M. aviumsubsp. paratuberculosis ΔsigL did not persist in the intestines as wewere unable to detect any bacteria (limit of detection 20 CFU) at 6 and12 WPI.

The histological analysis revealed mild to moderate granulomatousinflammation in the liver tissues at both 3 and 6 WPI with either of theM. avium subsp. paratuberculosis strains with higher lymphocyticinfiltration in the mice infected with M. avium subsp. paratuberculosisΔsigL. At 12 WPI, M. avium subsp. paratuberculosis ΔsigL infectedanimals showed less granulomatous inflammation indicating reducedability of M. avium subsp. paratuberculosis ΔsigL compared withwild-type bacilli to establish an infection in animals. In accordancewith the bacterial organ burden data, Ziehl-Neelsen staining showedhigher numbers of acid-fast bacilli in mice liver infected with thewild-type strain relative to the M. avium subsp. paratuberculosis ΔsigLat all examined time points. A similar observation was noticed for thespleen and intestine tissues (data not shown). Both bacterial organcolonization and histological data analyses suggested that M. aviumsubsp. paratuberculosis ΔsigL was attenuated for survival, compared tothe wild-type strain in the mice model of infection.

Immunization with M. avium subsp. paratuberculosis ΔsigL. Because sigLencodes a mycobacterial GGR (32) and was critical for M. avium subsp.paratuberculosis survival in the present study study, we investigatedthe vaccine potential of M. avium subsp. paratuberculosis ΔsigL in achallenge model of murine paratuberculosis (FIG. 5A). To examineimmunogenicity of the mutant strain, groups of mice were immunized twicewith M. avium subsp. paratuberculosis ΔsigL and 6 weekspost-immunization (WPI) mice organs were analyzed for bacterial content.Two immunizations with this mutant resulted in low colonization (2×10²CFU) in the liver whereas no bacteria were detected (limit of detection20 CFU) in the intestine or spleen (data not shown). To evaluate vaccineinduced immune responses before challenge; ELISA was used to estimatelevels of key cytokines in stimulated spleen cells. Statistical analysisrevealed significantly (p<0.05) high level of IFN-γ secretion in the M.avium subsp. paratuberculosis ΔsigL immunized mice compared to naïveanimals (FIG. 5B). Because of the importance of T-helper 17 cells (33)for intracellular bacterial infection, we examined IL-17A production inthe immunized animals. However, we did not find any detectable levelsfor IL-17A at 6 WPI. Additionally, the mice group vaccinated with M.avium subsp. paratuberculosis ΔsigL had significantly (p<0.05) higheranti-PPDj IgG level (FIG. 5C). Thus, both IFN-γ and IgG data suggestedability of the M. avium subsp. paratuberculosis ΔsigL strain to induceenhanced immune responses.

Protection against challenge with M. avium subsp. paratuberculosis. Toexamine the vaccine potential of sigL-based mutant, groups of mice werevaccinated with PBS (control) or M. avium subsp. paratuberculosis ΔsigLlive strain and challenged with the virulent M. avium subsp.paratuberculosis K10 strain following 6 WPI (FIG. 5A). At 6 weeks postchallenge (WPC), the ΔsigL mice group had a significant reduction in thebacterial load in spleen and liver (˜0.5 log) compared to thePBS-vaccinated mice (FIG. 6A, B). More importantly, a higher level ofbacterial load reduction (˜1 log) was observed in the intestine (FIG.6C), an important organ for M. avium subsp. paratuberculosis infection.A similar colonization pattern was observed for all examined organs(spleen, liver, intestine) at 12 WPC where the level of bacterialreduction reached >1 log.

For histological examination, we focused our efforts on the liverbecause it is the most reflective organ for M. avium subsp.paratuberculosis infection (34). Liver sections from ΔsigL immunizedanimals displayed lower granulomatous scores and smaller size granulomasthan the PBS-control group at 6 and 12 WPC (FIG. 7A-B). In addition, lownumbers of acid-fast bacilli were observed when liver sections werestained with Ziehl-Neelsen stain, another support for colonization datadiscussed above. Interestingly, sections from the intestines of theΔsigL-immunized mice appeared normal compared to mock infected mice withno detectable acid-fast bacteria in Ziehl-Neelsen stained sections atboth at 6 and 12 WPC (FIG. 7C-D). Overall, reduction in M. avium subsp.paratuberculosis colonization levels combined with histological scoresindicated the ability of the ΔsigL mutant to control tissue damage by achallenge with the virulent strain of M. avium subsp. paratuberculosis.

Expansion of immune responses following challenge in immunized mice. Toevaluate expansion of the cellular immune response following challenge,splenocytes of immunized and challenged mice were analyzed for theproduction of key cytokines associated with protection againstparatuberculosis (35,36). As shown in FIG. 6D, PPD-stimulatedsplenocytes from ΔsigL-immunized and challenged mice secretedsignificantly higher levels of IFN-γ than that of control animals at6WPC, indicating increased levels of T cell activity (T-helper 1 cells)in the animals that received ΔsigL mutant. However, at 12 WPC there wasno significant difference in IFN-γ response between these two groups ofanimals. Our data also showed a better ability, even though notsignificant, of the M. avium subsp. paratuberculosis ΔsigL vaccinatedanimals to induce PPD-specific IL-17A secretion compared to the mockchallenged group at 6WPC. Taken together, the colonization, histologicaland immune response levels suggested that M. avium subsp.paratuberculosis ΔsigL induced protective immunity against challengewith virulent M. avium subsp. paratuberculosis strain.

Discussion

Infection with M. avium subsp. paratuberculosis represent a major threat(Johne's disease) to dairy animals (11,14) with the potential spread tohuman with the likely involvement in several diseases (7-10,12). Earlierreports indicated that M. avium subsp. paratuberculosis count on a largenumber of sigma factors (N=19) to establish the infection and survivediverse stress conditions (19,37). In this study, we have targeted sigLbecause of its activation during early macrophage infection suggesting arole to control important stage(s) of M. avium subsp. paratuberculosispathogenesis following oral infection (19). Moreover, the orthologoussigL deletion mutant, M. tuberculosis ΔsigL, in M. tuberculosis was lesslethal for mice relative to the wild-type strain (32). However, unlikeM. tuberculosis ΔsigL (24), our analysis indicated that deletion of sigLaffected ability of M. avium subsp. paratuberculosis to survive exposureto intracellular stimuli including oxidative stress and damaging themycobacterial cell wall stresses (e.g. diamide and SDS) (38-40). Thismutant was also unable to survive both naïve and activated macrophagesto similar levels, an indication of the significant attenuation of thismutant. This intracellular survival defect of the mutant was furtherverified by the poor ability of M. avium subsp. paratuberculosis ΔsigLto replicate in mouse tissues. Both histological and bacteriologicalanalyses revealed reduced organ colonization of the M. avium subsp.paratuberculosis ΔsigL with low inflammatory scores compared to theparental strain. Interestingly, this result was in contrast to thereports where orthologous M. tuberculosis ΔsigL survived in the miceorgans to the same level as the wild-type strain suggesting a morecomprehensive and dynamic role for sigL in M. avium subsp.paratuberculosis survival and pathogenesis (24,32). However, the natureand mechanisms employed by sigL to enable M. avium subsp.paratuberculosis virulence remain elusive.

The alternative sigma factor (e.g. sigE) mutant strains were targetedfor the vaccine development and found to provide protection againstinfection with pathogenic bacteria including mycobacteria (41,42). Inour study, observations gained from both in vitro and murine modelexperiments encouraged us to investigate the vaccine potential of M.avium subsp. paratuberculosis ΔsigL as a live attenuated vaccine againstM. avium subsp. paratuberculosis infection in mice. The murine model ofparatuberculosis represent an important screening tool (43,44) toexamine vaccine candidates before testing in a more expensive goatmodel, despite the associated shortcomings of the murine model (nodiarrhea or shedding of bacteria). In our hands, mice that received M.avium subsp. paratuberculosis ΔsigL were very efficient in producingIFN-γ (e.g. 6WPI), an important cytokine involved in controllingmycobacterial infection (45). The decline in IFN-γ is often associatedwith the onset of clinical JD in ruminants (35,46). Importantly,culturing tissue samples from immunized animals indicated the ability ofM. avium subsp. paratuberculosis ΔsigL to persist in animals followingimmunization but to a low level which could be a critical factor ininducing protective immune responses.

Controlling M. avium subsp. paratuberculosis infection is dependent ondeveloping vaccines that reduce bacterial colonization and shedding frominfected animals. Earlier studies demonstrated the potential use of M.avium subsp. paratuberculosis mutants (e.g. WAg915, M. avium subsp.paratuberculosis AleuD) as live attenuated vaccine candidates in themurine model of paratuberculosis (44,47). WAg915 mutant strain (M. aviumsubsp. paratuberculosis AppiA), defective in the peptidyl-prolylcis-trans-isomerase, showed mild attenuated phenotype relative to thewild-type strain and provided limited protection in mouse organ only atlater stages after challenge with parental M. avium subsp.paratuberculosis strain (47), whereas the attenuated M. avium subsp.paratuberculosis ΔleuD, defective in leucine biosynthesis, exhibitedsome protection following challenge (44). In our experiment, M. aviumsubsp. paratuberculosis ΔsigL was attenuated in macrophages and in micebut persisted in murine tissues up to the time of challenge. Ability topersist following immunization could be responsible for the generatedprotective immunity, which was more efficient to reduce M. avium subsp.paratuberculosis colonization compared to the Wag915 candidate (47).However, it is not clear whether M. avium subsp. paratuberculosis ΔsigLcould provide better protection compared to the M. avium subsp.paratuberculosis AleuD because the previous study examined M. aviumsubsp. paratuberculosis bacilli in mouse tissues by Ziehl-Neelsenstaining which is less sensitive compared to the tissue culturing(48,49). We further evaluated longevity of immune responses in the mousegroups following challenge and these data suggest that M. avium subsp.paratuberculosis ΔsigL immunized mice maintained strong T-cell responseswith secretion of higher IFN-γ and IL-17 at 6WPC, despite the later notbeing significant compared to the mock infected group.

Overall, an isogenic mutant of M. avium subsp. paratuberculosis lackingsigL had limited ability to survive in macrophages or mice, most likelybecause of a defective bacterial cell wall. Such an attenuated strain ofM. avium subsp. paratuberculosis (ΔsigL) persisted in murine tissuefollowing subcutaneous immunization and generated a robust immuneresponse. The generated immune responses were sufficient to reducetissue colonization and lesion scores in animals following a challengewith the wild-type strain of M. avium subsp. paratuberculosis. Furthervaccine testing in natural hosts of Johne's disease, e.g. goats orcalves, will demonstrate the viability of developing an effectivecontrol strategy against paratuberculosis in both animals and humans. Ingeneral, approaches used to investigate sigL in M. avium subsp.paratuberculosis could be adopted to examine the role and potential useof other global gene regulators in pathogenesis and control ofintracellular pathogens.

Example 3

References cited in this Example are listed in the section of Referencesas “References cited in Example 3.”

Survival of GGR mutants in the murine model of paratuberculosis. Toassess the role of sigH and sigL in M. ap virulence, we investigated thepersistence of the each mutant in mice. The initial growth kinetic ofthe wild-type and ΔsigH mutant strains was similar, with an equal burdenof bacteria in liver, intestine and spleen up to 6 WPI (FIG. 8).However, at 12 WPI, colonization levels were significantly reduced forΔsigH compared to its parental strain only in spleen and intestine(p<0.01 and p<0.05, respectively), suggesting a role for sigH in thelong term survival of M. ap in mice. For the ΔsigL mutant, bacterialcolonization levels were significantly reduced in spleen, liver andintestine at all times post infection (FIG. 8) compared to K10,suggesting a pivotal role for sigL in the pathogenesis of M. ap.

Interestingly, when the ΔsigH mutant of M. tuberculosis was used tochallenge mice, no differences in bacterial load were observed in mouseorgans compared to the wild-type strain (54), suggesting a moresignificant role for sigH in M. ap. Consistent with the colonizationdata, Ziehl-Neelsen staining of spleen showed higher acid fast bacilliin mice infected with wild-type strain compared to ΔsigH mutant at 12WPI (FIG. 9).

Taken together, our data indicated that both ΔsigH and ΔsigL mutantswere attenuated in the murine model of paratuberculosis compared to thewild-type M. ap strain.

RNA sequencing for transcriptional profiling. During the last decade, itis evident that the use of DNA microarrays has greatly contributed toour understanding of the genetic basis of bacterial infections,including infection with M. ap (42,55-58). Recently, large scalesequencing approaches were developed for gene expression profiling toovercome problems associated with DNA microarrays (59). The use ofNext-generation sequencers enabled us to sequence all transcripts in agiven RNA sample, hence named RNA sequence (RNA-Seq) profiling. Thisnaming reflects the ability of the high throughput sequencers (e.g.HighSeq2000, Roche 454) to be used for in-depth sequencing of millionsof transcripts in a single run (59-61). The RNA-Seq approach has alreadybeen successfully applied in several systems, both mammalian andbacterial, (61) including M. tuberculosis and M. ap in our laboratory(62) (see below). Some key benefits of RNA-Seq over microarrays includeless biased transcriptome data acquisition, actual sequences fortranscripts that can show SNPs and antisense transcription, less complexdata analysis; improved correlation between laboratories and increasedsensitivity of low transcript numbers (63-65).

Because of the global nature of transcriptional regulation under controlof a factors, we pursued the characterization of the sigH transcriptomeusing RNA-Seq profiling utilizing an Illumina-based sequencer operatedby the University of Wisconsin Biotechnology Center (UWBC). Our stressexperiments showed that the ΔsigH mutant was hypersensitive to elevatedtemperature and diamide exposure, each resulting in impaired growth.Accordingly, we hypothesize that sigH may play an important role indirecting transcriptional control under unfavorable environmentalconditions. To test this hypothesis, both wild-type M. ap K10 and ΔsigHmutant transcriptomes were profiled before and after diamide exposure.Briefly, cultures grown to mid-log phase (OD₆₀₀=0.5) from both K-10 andits isogenic mutant, ΔsigH, were centrifuged (3,200×g) for 15 min at 4°C. for total RNA extraction as outlined before (32). Extracted RNAsamples were treated with DNAse I (Ambion) to eliminate residual genomicDNA and then treated with MICROBExpress (Ambion, Austin) to enrichbacterial mRNA and reduce the amount of rRNA. Following a standardprotocol for cDNA generation in our laboratory (42), all samples weresent to the UWBC for library construction and sequencing on theIllumina/GAIIx sequencing platform using 100-bp, paired end fragments.As a testament of the high resolution analysis of RNA-Seq profiling, wewere not able to detect the presence of only 18 or 20 genes in bothsamples from M. ap K10 or ΔsigH, respectively (Table 2).

TABLE 1 Sequencing run statistics on M. ap samples. M. ap K10 M. ap K10ΔsigH Material Submitted 2 μg mRNA 2 μg mRNA Total Reads 74,747,58253,305,460 Read Length 100 bp 100 bp Total Reads Mapped 57,477,92041,101,069 Reads mapped in pairs 53,359,286 36,887,226 Reads mapped to54,209,373 37,081,401 rRNA or tRNA Reads not mapped 16,903,46411,874,445 Most sequenced MAP1975 MAP1975 mRNA (reads) (1,286,560)(1,390,707) Transcripts not 18 20 detected

Examine the vaccine potential of GGR M. ap mutants. Because they controla large number of genes, we hypothesize that GGR mutants will beattenuated but able generate enough immune responses to serve as liveattenuated vaccines that could control JD. To examine the vaccinepotential of ΔsigH and ΔsigL mutants as live-attenuated vaccines,C57BL/6 mice groups (N=11) were vaccinated twice at 2 weeks intervalwith 10⁶ CFU/mouse via subcutaneous (S/C.) injection. This route ofimmunization was chosen after evaluating other routes (oral andintra-peritoneal, I/P.) and was found to yield better immunity (data notshown). In addition, immunized mice via the S/C. showed no M. apcolonization in their organs when examined at 6 weeks post immunization(WPI). At 6 WPI, immunized mice (N=8/group) were challenged with 2×10⁸CFU/mouse of the wild-type strain M. ap K10 via I/P. Mock-immunized micewith (Phosphate Buffered Saline, PBS) showed the highest colonizationlevels of M. ap in their organs following challenge with M. ap K10. Onthe other hand, mice vaccinated with the attenuated mutants (ΔsigH andΔsigL) showed a significant reduction in bacterial colony levels in theintestine at 6 weeks post challenge (WPC) (FIG. 10). OnlyΔsigL-vaccinated group showed significant reduction in both liver andspleen at this time point. By 12 WPC, only ΔsigL-vaccinated group showedmore than 1 log reduction in intestine in all organs (intestine, liver,spleen) compared to the control group (FIG. 10). Overall, ΔsigLimmunization was more effective in inducing protection against virulentM. ap K10 strain compared to ΔsigH vaccination.

REFERENCES

1. Ghosh, P., C. Hsu, E. J. Alyamani, M. M. Shehata, M. A. Al-Dubaib, A.Al-Naeem, M. Hashad, O. M. Mahmoud, K. B. Alharbi, K. Al-Busadah, A. M.Al-Swailem, and A. M. Talaat. 2012. Genome-wide analysis of the emerginginfection with Mycobacterium avium subspecies paratuberculosis in theArabian camels (Camelus dromedarius). PLoS. One. 7:e31947.doi:10.1371/journal.pone.0031947 [doi]; PONE-D-11-17590 [pii].

2. Yalo Ayele, W., M. Machackova, and I. Pavlik. 2001. The transmissionand impact of paratuberculosis infection in domestic and wild ruminants.Vet. Med.(Praha) 46:205-224.

3. Nielsen, S. r. S. and N. Toft. 2009. A review of prevalences ofparatuberculosis in farmed animals in Europe. Preventive VeterinaryMedicine 88:1-14. doi:doi: DOI: 10.1016/j.prevetmed.2008.07.003.

4. Nielsen, S. S. and Kennedy, D. Proceedings of the 1st ParaTB Forum.Nielsen, S. S. and Kennedy, D. The 1st ParaTB Forum. Proceedings of the1st ParaTB Forum, 1-48. Mar. 22, 2007. Brussels, Belgium, InternationalDairy Federation.

5. National Animal Health Monitoring System. 2008. Johne's Disease onU.S. Dairies, 1991-2007. USDA-APHIS Veterinary Services, Ft. Collins,Colo. 2008.

6. Ott, S. L., S. J. Wells, and B. A. Wagner. 1999. Herd-level economiclosses associated with Johne's disease on US dairy operations. Prev.Vet. Med. 40:179-192. doi:S0167-5877 (99)00037-9 [pii].

7. NIAA. The cost of Johne's disease to dairy producersNationalInstitute for Animal Agriculture. Springs, Colo. (CO) (2009)09-69224-00. 2009.

8. Bermudez, L. E., M. Petrofsky, S. Sommer, and R. G. Barletta. 2010.Peyer's patch 684 deficient mice demonstrate that Mycobacterium aviumsubsp. Paratuberculosis translocates across the mucosal barrier via bothM cells and enterocytes but has inefficient dissemination. Infect.Immun. 78:3570-3577. doi:IAI.01411-09 [pii]; 10.1128/IAI.01411-09 [doi].

9. Ponnusamy, D., S. Periasamy, B. N. Tripathi, and A. Pal. 2012.Mycobacterium avium subsp. paratuberculosis invades through M cells andenterocytes across ileal and jejuna mucosa of lambs. Res. Vet. Sci. doi:S0034-5288 (12)00301-3 [pii]; 10.1016/j.rvsc.2012.09.023 [doi].

10. Chacon, O., L. E. Bermudez, and R. G. Barletta. 2004. Johne'sdisease, inflammatory bowel disease, and Mycobacterium paratuberculosis.Annu. Rev. Microbiol. 58:329-363.

11. Coussens, P. M., C. J. Colvin, K. Wiersma, A. Abouzied, and S.Sipkovsky. 2002. Gene expression profiling of peripheral bloodmononuclear cells from cattle infected with Mycobacteriumparatuberculosis. Infect. Immun. 70:5494-5502.

12. Coussens, P. M., A. Jeffers, and C. Colvin. 2004. Rapid andtransient activation of gene expression in peripheral blood mononuclearcells from Johne's disease positive cows exposed to Mycobacteriumparatuberculosis in vitro. Microbial. pathogenesis. 36:93-108.

13. Weiss, D. J., O. A. Evanson, M. Deng, and M. S. Abrahamsen. 2004.Sequential patterns of gene expression by bovine monocyte-derivedmacrophages associated with ingestion of mycobacterial organisms.Microb. Pathog. 37:215-224.

14. Weiss, D. J., O. A. Evanson, M. Deng, and M. S. Abrahamsen. 2004.Gene expression and antimicrobial activity of bovine macrophages inresponse to Mycobacterium avium subsp. paratuberculosis. Vet. Pathol.41:326-337.

15. Wu, C. W., S. K. Schmoller, S. J. Shin, and A. M. Talaat. 2007.Defining the stressome of Mycobacterium avium subsp paratuberculosis invitro and in naturally infected cows. Journal of Bacteriology189:7877-7886.

16. Whittington, R. J., D. J. Marshall, P. J. Nicholls, I. B. Marsh, andL. A. Reddacliff. 2004. Survival and dormancy of Mycobacterium aviumsubsp. paratuberculosis in the environment. Appl. Environ. Microbiol.70:2989-3004.

17. Bannantine, J. P. and J. R. Stabel. 2002. Killing of Mycobacteriumavium subspecies paratuberculosis within macrophages. BMC Microbiol.2:2.

18. Stabel, J. R., M. V. Palmer, B. Harris, B. Plattner, J. Hostetter,and S. Robbe-Austerman. 2009. Pathogenesis of Mycobacterium avium subsp.paratuberculosis in neonatal calves after oral or intraperitonealexperimental infection. Vet. Microbiol. 136:306-313.

19. Wu, C. W., M. Livesey, S. K. Schmoller, E. J. Manning, H. Steinberg,W. C. Davis, M. J. Hamilton, and A. M. Talaat. 2007. Invasion andpersistence of Mycobacterium avium subsp. paratuberculosis during earlystages of Johne's disease in calves. Infect. Immun. 75:2110-2119.

20. Shin, S. J., C.-W. Wu, H. Steinberg, and A. M. Talaat. 2006.Identification of Novel Virulence Determinants in Mycobacteriumparatuberculosis by Screening a Library of Insertional Mutants. Infec.Immun. 7:3825-3833.

21. Alonso-Hearn, M., D. Patel, L. Danelishvili, L. Meunier-Goddik, andL. E. Bermudez. 2008. The Mycobacterium avium subsp. paratuberculosisMAP3464 gene encodes an oxidoreductase involved in invasion of bovineepithelial cells through the activation of host cell Cdc42. Infect.Immun. 76:170-178.

22. Wu, C. W., S. K. Schmoller, J. P. Bannantine, T. M. Eckstein, J. M.Inamine, M. Livesey, R. Albrecht, and A. M. Talaat. 2009. A novel cellwall lipopeptide is important for biofilm formation and pathogenicity ofMycobacterium avium subspecies paratuberculosis. Microb. Pathog.46:222-230.

23. Alonso-Hearn, M., T. M. Eckstein, S. Sommer, and L. E. Bermudez.2010. A Mycobacterium avium subsp. paratuberculosis LuxR regulates cellenvelope and virulence. Innate immunity 16:235-247.

24. Zhu, X., Z. J. Tu, P. M. Coussens, V. Kapur, H. Janagama, S. Naser,and S. Sreevatsan. 2008. Transcriptional analysis of diverse strainsMycobacterium avium subspecies paratuberculosis in primary bovinemonocyte derived macrophages. Microbes Infect. 10:1274-1282.

25. Janagama, H. K., E. A. Lamont, S. George, J. P. Bannantine, W. W.Xu, Z. J. Tu, S. J. Wells, J. Schefers, and S. Sreevatsan. 2010. Primarytranscriptomes of Mycobacterium avium subsp. paratuberculosis revealproprietary pathways in tissue and macrophages. BMC Genomics 11:561.

26. Manganelli, R., M. I. Voskuil, G. K. Schoolnik, E. Dubnau, M. Gomez,and I. Smith. 2002. Role of the extracytoplasmic-function sigma Factorsigma(H) in Mycobacterium tuberculosis global gene expression. Mol.Microbiol. 45:365-374.

27. Paget, M. S., J. G. Kang, J. H. Roe, and M. J. Buttner. 1998.sigmaR, an RNA polymerase sigma factor that modulates expression of thethioredoxin system in response to oxidative stress in Streptomycescoelicolor A3 (2). EMBO J. 17:5776-5782. doi:10.1093/emboj/17.19.5776[doi].

REFERENCES CITED IN EXAMPLE 1

1. Ghosh, P., C. Hsu, E. J. Alyamani, M. M. Shehata, M. A. Al-Dubaib, A.Al-Naeem, M. Hashad, O. M. Mahmoud, K. B. Alharbi, K. Al-Busadah, A. M.Al-Swailem, and A. M. Talaat. 2012. Genome-wide analysis of the emerginginfection with Mycobacterium avium subspecies paratuberculosis in theArabian camels (Camelus dromedarius). PLoS.One. 7:e31947.

2. Yalo Ayele, W., M. Machackova, and I. Pavlik. 2001. The transmissionand impact of paratuberculosis infection in domestic and wild ruminants.Vet. Med. 46:205-224.

3. Nielsen, S. r. S. and N. Toft. 2009. A review of prevalences ofparatuberculosis in farmed animals in Europe. Preventive VeterinaryMedicine 88:1-14.

4. Lombard, J. E. 2011. Epidemiology and economics of paratuberculosis.Vet. Clin. North Am. Food Anim Pract. 27:525-35.

5. Lombard, J. E., I. A. Gardner, S. R. Jafarzadeh, C. P. Fossler, B.Harris, R. T. Capsel, B. A. Wagner, and W. O. Johnson. 2013. Herd-levelprevalence of Mycobacterium avium subsp. paratuberculosis infection inUnited States dairy herds in 2007. Prev. Vet. Med. 108:234-238.

6. Ott, S. L., S. J. Wells, and B. A. Wagner. 1999. Herd-level economiclosses associated with Johne's disease on US dairy operations. Prev.Vet. Med. 40:179-192.

7. Bermudez, L. E., M. Petrofsky, S. Sommer, and R. G. Barletta. 2010.Peyer's patch-deficient mice demonstrate that Mycobacterium avium subsp.paratuberculosis translocates across the mucosal barrier via both Mcells and enterocytes but has inefficient dissemination. Infect. Immun.78:3570-3577.

8. Chacon, O., L. E. Bermudez, and R. G. Barletta. 2004. Johne'sdisease, inflammatory bowel disease, and Mycobacterium paratuberculosis.Annu. Rev. Microbiol. 58:329-363.

9. Coussens, P. M., C. J. Colvin, K. Wiersma, A. Abouzied, and S.Sipkovsky. 2002. Gene expression profiling of peripheral bloodmononuclear cells from cattle infected with Mycobacteriumparatuberculosis. Infect. Immun. 70:5494-5502.

10. Coussens, P. M., A. Jeffers, and C. Colvin. 2004. Rapid andtransient activation of gene expression in peripheral blood mononuclearcells from Johne's disease positive cows exposed to Mycobacteriumparatuberculosis in vitro. Microbial. pathogenesis. 36:93-108.

11. Weiss, D. J., O. A. Evanson, M. Deng, and M. S. Abrahamsen. 2004.Gene expression and antimicrobial activity of bovine macrophages inresponse to Mycobacterium avium subsp. paratuberculosis. Vet. Pathol.41:326-337.

12. Wu, C. W., S. K. Schmoller, S. J. Shin, and A. M. Talaat. 2007.Defining the stressome of Mycobacterium avium subsp paratuberculosis invitro and in naturally infected cows. Journal of Bacteriology189:7877-7886.

13. Whittington, R. J., D. J. Marshall, P. J. Nicholls, I. B. Marsh, andL. A. Reddacliff. 2004. Survival and dormancy of Mycobacterium aviumsubsp. paratuberculosis in the environment. Appl. Environ. Microbiol.70:2989-3004.

14. Bannantine, J. P. and J. R. Stabel. 2002. Killing of Mycobacteriumavium subspecies paratuberculosis within macrophages. BMC Microbiol.2:2.

15. Stabel, J. R., M. V. Palmer, B. Harris, B. Plattner, J. Hostetter,and S. Robbe-Austerman. 2009. Pathogenesis of Mycobacterium avium subsp.paratuberculosis in neonatal calves after oral or intraperitonealexperimental infection. Vet. Microbiol. 136:306-313.

16. Wu, C. W., M. Livesey, S. K. Schmoller, E. J. Manning, H. Steinberg,W. C. Davis, M. J. Hamilton, and A. M. Talaat. 2007. Invasion andpersistence of Mycobacterium avium subsp. paratuberculosis during earlystages of Johne's disease in calves. Infect. Immun. 75:2110-2119.

17. Shin, S. J., C.-W. Wu, H. Steinberg, and A. M. Talaat. 2006.Identification of Novel Virulence Determinants in Mycobacteriumparatuberculosis by Screening a Library of Insertional Mutants. Infec.Immun. 7:3825-3833.

18. Alonso-Hearn, M., D. Patel, L. Danelishvili, L. Meunier-Goddik, andL. E. Bermudez. 2008. The Mycobacterium avium subsp. paratuberculosisMAP3464 gene encodes an oxidoreductase involved in invasion of bovineepithelial cells through the activation of host cell Cdc42. Infect.Immun. 76:170-178.

19. Wu, C. W., S. K. Schmoller, J. P. Bannantine, T. M. Eckstein, J. M.Inamine, M. Livesey, R. Albrecht, and A. M. Talaat. 2009. A novel cellwall lipopeptide is important for biofilm formation and pathogenicity ofMycobacterium avium subspecies paratuberculosis. Microb. Pathog.46:222-230.

20. Alonso-Hearn, M., T. M. Eckstein, S. Sommer, and L. E. Bermudez.2010. A Mycobacterium avium subsp. paratuberculosis LuxR regulates cellenvelope and virulence. Innate immunity 16:235-247.

21. Zhu, X., Z. J. Tu, P. M. Coussens, V. Kapur, H. Janagama, S. Naser,and S. Sreevatsan. 2008. Transcriptional analysis of diverse strainsMycobacterium avium subspecies paratuberculosis in primary bovinemonocyte derived macrophages. Microbes. Infect. 10:1274-1282.

22. Janagama, H. K., E. A. Lamont, S. George, J. P. Bannantine, W. W.Xu, Z. J. Tu, S. J. Wells, J. Schefers, and S. Sreevatsan. 2010. Primarytranscriptomes of Mycobacterium avium subspecies paratuberculosis revealproprietary pathways in tissue and macrophages. BMC Genomics.11:561.:561.

23. Arsenault, R. J., Y. Li, K. Bell, K. Doig, A. Potter, P. J. Griebel,A. Kusalik, and S. Napper. 2012. Mycobacterium avium subspeciesparatuberculosis Inhibits Gamma Interferon-Induced Signaling in BovineMonocytes: Insights into the Cellular Mechanisms of Johne's Disease.Infect. & Immun. 80:3039-3048.

24. Sechi, L. A., G. E. Felis, N. Ahmed, D. Paccagnini, D. Usai, S.Ortu, P. Molicotti, and S. Zanetti. 2007. Genome and transcriptome scaleportrait of sigma factors in Mycobacterium avium subsp.paratuberculosis. Infection, Genetics and Evolution 7:424-432.

25. Raman, S., T. Song, X. Puyang, S. Bardarov, W. R. Jacobs, Jr., andR. N. Husson. 2001. The Alternative Sigma Factor SigH Regulates MajorComponents of Oxidative and Heat Stress Responses in Mycobacteriumtuberculosis. The Journal of Bacteriology 183:6119-6125.

26. Diwu, Z., C. S. Chen, C. Zhang, D. H. Klaubert, and R. P. Haugland.1999. A novel acidotropic pH indicator and its potential application inlabeling acidic organelles of live cells. Chemistry & biology 6:411-418.

27. Wozniak, A. L., S. Griffin, D. Rowlands, M. Harris, M. Yi, S. M.Lemon, and S. A. Weinman. 2010. Intracellular proton conductance of thehepatitis C virus p7 protein and its contribution to infectious virusproduction. PLoS Pathog. 6:e1001087.

28. Abramoff, M. D., P. J. Magelhaes, and S. J. Ram. 2004. ImageProcessing with ImageJ. Biophotonics International 11:36-42.

29. Grode, L., P. Seiler, S. Baumann, J. Hess, V. Brinkmann, E. A.Nasser, P. Mann, C. Goosmann, S. Bandermann, D. Smith, G. J. Bancroft,J. M. Reyrat, S. D. van, B. Raupach, and S. H. Kaufmann. 2005. Increasedvaccine efficacy against tuberculosis of recombinant Mycobacterium bovisbacille Calmette-Guerin mutants that secrete listeriolysin. The Journalof clinical investigation 115:2472-2479.

30. Rohde, K. H., R. B. Abramovitch, and D. G. Russell. 2007.Mycobacterium tuberculosis invasion of macrophages: linking bacterialgene expression to environmental cues. Cell Host Microbe 2:352-364.

31. Talaat, A. M., S. T. Howard, W. Hale, R. Lyons, H. Garner, and S. A.Johnston. 2002. Genomic DNA standards for gene expression profiling inMycobacterium tuberculosis. Nucleic Acids Res. 30:e104.

32. Li, L., J. P. Bannantine, Q. Zhang, A. Amonsin, B. J. May, D. Alt,N. Banerji, S. Kanjilal, and V. Kapur. 2005. The complete genomesequence of Mycobacterium avium subspecies paratuberculosis. Proc. Natl.Acad. Sci U.S.A 102:12344-12349.

33. Talaat, A. M., P. Hunter, and S. A. Johnston. 2000. Genome-directedprimers for selective labeling of bacterial transcripts for DNAmicroarray analysis. Nat. Biotechnol. 18:679-682.

34. Raychaudhuri, S., J. M. Stuart, and R. B. Altman. 2000. Principalcomponents analysis to summarize microarray experiments: application tosporulation time series. Pac. Symp. Biocomput. 455-466.

35. Wynne, J. W., T. Seemann, D. M. Bulach, S. A. Coutts, A. M. Talaat,and W. P. Michalski. 2010. Resequencing the Mycobacterium avium subsp.paratuberculosis K10 genome: improved annotation and revised genomesequence. J. Bacteriol. 192:6319-6320.

36. Mortazavi, A., B. A. Williams, K. McCue, L. Schaeffer, and B. Wold.2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat.Methods 5:621-628.

37. Kal, A. J., A. J. van Zonneveld, V. Benes, M. van den Berg, M. G.Koerkamp, K. Albermann, N. Strack, J. M. Ruijter, A. Richter, B. Dujon,W. Ansorge, and H. F. Tabak. 1999. Dynamics of gene expression revealedby comparison of serial analysis of gene expression transcript profilesfrom yeast grown on two different carbon sources. Mol. Biol. Cell10:1859-1872.

38. Edgar, R., M. Domrachev, and A. E. Lash. 2002. Gene ExpressionOmnibus: NCBI gene expression and hybridization array data repository.Nucleic Acids Res. 30:207-210.

39. Ward, S. K., E. A. Hoye, and A. M. Talaat. 2008. The globalresponses of Mycobacterium tuberculosis to physiological levels ofcopper. J. Bacteriol. 190:2939-2946.

40. Talaat, A. M., S. K. Ward, C.-W. Wu, E. Rondon, C. Tavano, J. P.Bannantine, R. Lyons, and S. A. Johnston. 2007. Mycobacterial bacilliare metabolically active during chronic tuberculosis in murine lungs:Insights from genome-wide transcriptional profiling. J. Bacteriol.189:4265-4274.

41. Pfaffl, M. W. 2001. A new mathematical model for relativequantification in real-time RT-PCR. Nucleic Acids Res. 29:e45.

42. Bannantine, J. P. and A. M. Talaat. 2010. Genomic and transcriptomicstudies in Mycobacterium avium subspecies paratuberculosis. Vet.Immunol. Immunopathol. 138:303-311.

43. Janagama, H. K., T. M. Senthilkumar, J. P. Bannantine, G. M.Rodriguez, I. Smith, M. L. Paustian, J. A. McGarvey, and S. Sreevatsan.2009. Identification and functional characterization of theiron-dependent regulator (IdeR) of Mycobacterium avium subsp.paratuberculosis. Microbiology 155:3683-3690.

44. Schnappinger, D., S. Ehrt, M. I. Voskuil, Y. Liu, J. A. Mangan, I.M. Monahan, G. Dolganov, B. Efron, P. D. Butcher, C. Nathan, and G. K.Schoolnik. 2003. Transcriptional Adaptation of Mycobacteriumtuberculosis within Macrophages: Insights into the PhagosomalEnvironment. J. Exp. Med. 198:693-704.

45. Pagan-Ramos, E., S. S. Master, C. L. Pritchett, R. Reimschuessel, M.Trucksis, G. S. Timmins, and V. Deretic. 2006. Molecular andphysiological effects of mycobacterial oxyR inactivation. J. Bacteriol.188:2674-2680.

46. den Hengst, C. D. and M. J. Buttner. 2008. Redox control inactinobacteria. Biochim. Biophys. Acta 1780:1201-1216.

47. Bashyam, M. D. and S. E. Hasnain. 2004. The extracytoplasmicfunction sigma factors: role in bacterial pathogenesis. Infect. Genet.Evol. 4:301-308.

48. Sachdeva, P., R. Misra, A. K. Tyagi, and Y. Singh. 2010. The sigmafactors of Mycobacterium tuberculosis: regulation of the regulators.Febs Journal 277:605-626.

49. Kazmierczak, M. J., M. Wiedmann, and K. J. Boor. 2005. Alternativesigma factors and their roles in bacterial virulence. Microbiol. Mol.Biol. Rev. 69:527-543.

50. Dubnau, E., P. Fontan, R. Manganelli, S. Soares-Appel, and I. Smith.2002. Mycobacterium tuberculosis genes induced during infection of humanmacrophages. Infect. Immun. 70:2787-2795.

51. Manganelli, R., R. Provvedi, S. Rodrigue, J. Beaucher, L. Gaudreau,and I. Smith. 2004. Sigma factors and global gene regulation inMycobacterium tuberculosis. J. Bacteriol. 186:895-902.

52. Graham, J. E. and J. E. Clark-Curtiss. 1999. Identification ofMycobacterium tuberculosis RNAs synthesized in response to phagocytosisby human macrophages by selective capture of transcribed sequences(SCOTS). Proc. Natl. Acad. Sci. U.S.A 96:11554-11559.

53. Bardarov, S., Bardarov Jr S Jr, J. M. Pavelka, Jr., V.Sambandamurthy, M. Larsen, J. Tufariello, J. Chan, G. Hatfull, and J. W.Jacobs, Jr. 2002. Specialized transduction: an efficient method forgenerating marked and unmarked targeted gene disruptions inMycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology148:3007-3017.

54. Song, T. S., S. L. Dove, K. H. Lee, and R. N. Husson. 2003. RshA, ananti-sigma factor that regulates the activity of the mycobacterialstress response sigma factor SigH. Molecular Microbiology 50:949-959.

55. Karls, R. K., J. Guarner, D. N. McMurray, K. A. Birkness, and F. D.Quinn. 2006. Examination of Mycobacterium tuberculosis sigma factormutants using low-dose aerosol infection of guinea pigs suggests a rolefor SigC in pathogenesis. Microbiology 152:1591-1600.

56. Raman, S., R. Hazra, C. C. Dascher, and R. N. Husson. 2004.Transcription regulation by the Mycobacterium tuberculosis alternativesigma factor SigD and its role in virulence. Journal of Bacteriology186:6605-6616.

57. Kaushal, D., B. G. Schroeder, S. Tyagi, T. Yoshimatsu, C. Scott, C.Ko, L. Carpenter, J. Mehrotra, Y. C. Manabe, R. D. Fleischmann, and W.R. Bishai. 2002. Reduced immunopathology and mortality despite tissuepersistence in a Mycobacterium tuberculosis mutant lacking alternativesigma factor, SigH. Proceedings of the National Academy of Sciences ofthe United States of America 99:8330-8335.

58. Echave, P., J. Tamarit, E. Cabiscol, and J. Ros. 2003. Novelantioxidant role of alcohol dehydrogenase E from Escherichia coli. J.Biol. Chem. 278:30193-30198.

59. Manganelli, R., M. I. Voskuil, G. K. Schoolnik, E. Dubnau, M. Gomez,and I. Smith. 2002. Role of the extracytoplasmic-function sigma Factorsigma(H) in Mycobacterium tuberculosis global gene expression. Mol.Microbiol. 45:365-374.

60. Singh, A., D. K. Crossman, D. Mai, L. Guidry, M. I. Voskuil, M. B.Renfrow, and A. J. Steyn. 2009. Mycobacterium tuberculosis WhiB3maintains redox homeostasis by regulating virulence lipid anabolism tomodulate macrophage response. PLoS. Pathog. 5:e1000545.

61. Cangelosi, G. A., J. S. Do, R. Freeman, J. G. Bennett, M. Semret,and M. A. Behr. 2006. The two-component regulatory system mtrAB isrequired for morphotypic multidrug resistance in Mycobacterium avium.Antimicrob. Agents Chemother. 50:461-468.

62. Zahrt, T. C. and V. Deretic. 2000. An essential two-component signaltransduction system in Mycobacterium tuberculosis. J. Bacteriol.182:3832-3838.

63. Mehra, S. and D. Kaushal. 2009. Functional genomics reveals extendedroles of the Mycobacterium tuberculosis stress response factor sigmaH.J. Bacteriol. 191:3965-3980.

64. Busche, T., R. Silar, M. Picmanova, M. Patek, and J. Kalinowski.2012. Transcriptional regulation of the operon encodingstress-responsive ECF sigma factor SigH and its anti-sigma factor RshA,and control of its regulatory network in Corynebacterium glutamicum.BMC. Genomics 13:445.

65. Paget, M. S., J. G. Kang, J. H. Roe, and M. J. Buttner. 1998.sigmaR, an RNA polymerase sigma factor that modulates expression of thethioredoxin system in response to oxidative stress in Streptomycescoelicolor A3 (2). EMBO J. 17:5776-5782.

66. Kuehnel, M. P., R. Goethe, A. Habermann, E. Mueller, M. Rohde, G.Griffiths, and P. Valentin-Weigand. 2001. Characterization of theintracellular survival of Mycobacterium avium ssp. paratuberculosis:phagosomal pH and fusogenicity in J774 macrophages compared with othermycobacteria. Cell Microbiol. 3:551-566.

67. Stewart, G. R., L. Wernisch, R. Stabler, J. A. Mangan, J. Hinds, K.G. Laing, D. B. Young, and P. D. Butcher. 2002. Dissection of theheat-shock response in Mycobacterium tuberculosis using mutants andmicroarrays. Microbiology 148:3129-3138.

68. Yang, H., S. Huang, H. Dai, Y. Gong, C. Zheng, and Z. Chang. 1999.The Mycobacterium tuberculosis small heat shock protein Hsp16.3 exposeshydrophobic surfaces at mild conditions: conformational flexibility andmolecular chaperone activity. Protein Sci. 8:174-179.

69. Zhao, B., M. T. Collins, and C. J. Czuprynski. 1997. Effects ofgamma interferon and nitric oxide on the interaction of Mycobacteriumavium subsp. paratuberculosis with bovine monocytes. Infect. Immun.65:1761-1766.

70. Basler, T., C. Brumshagen, A. Beineke, R. Goethe, and W. Baumer.2013. Mycobacterium avium subspecies impair dendritic cell maturation.Innate. Immun. (Epub ahead of print).

71. Kabara, E. and P. M. Coussens. 2012. Infection of Primary BovineMacrophages with Mycobacterium avium Subspecies paratuberculosisSuppresses Host Cell Apoptosis. Front Microbiol. 3:215.

72. Mehra, S., N. A. Golden, K. Stuckey, P. J. Didier, L. A. Doyle, K.E. Russell-Lodrigue, C. Sugimoto, A. Hasegawa, S. K. Sivasubramani, C.J. Roy, X. Alvarez, M. J. Kuroda, J. L. Blanchard, A. A. Lackner, and D.Kaushal. 2012. The Mycobacterium tuberculosis stress response factorSigH is required for bacterial burden as well as immunopathology inprimate lungs. J. Infect. Dis. 205:1203-1213.

REFERENCES CITED IN EXAMPLE 2

1. Nielsen, S. r. S. and N. Toft. 2009. A review of prevalences ofparatuberculosis in farmed animals in Europe. Preventive VeterinaryMedicine 88:1-14. doi:doi: DOI: 10.1016/j.prevetmed.2008.07.003.

2. Ghosh, P., C. Hsu, E. J. Alyamani, M. M. Shehata, M. A. Al-Dubaib, A.Al-Naeem, M. Hashad, O. M. Mahmoud, K. B. Alharbi, K. Al-Busadah, A. M.Al-Swailem, and A. M. Talaat. 2012. Genome-wide analysis of the emerginginfection with Mycobacterium avium subspecies paratuberculosis in theArabian camels (Camelus dromedarius). PLoS.One. 7:e31947.

3. Kumthekar, S., E. J. Manning, P. Ghosh, K. Tiwari, R. N. Sharma, andH. Hariharan. 2013. Mycobacterium avium subspecies paratuberculosisconfirmed following serological surveillance of small ruminants inGrenada, West Indies. J. Vet. Diagn. Invest 25:527-530.doi:1040638713490688 [pii]; 10.1177/1040638713490688 [doi].

4. McClure, H. M., R. J. Chiodini, D. C. Anderson, R. B. Swenson, W. R.Thayer, and J. A. Coutu. 1987. Mycobacterium paratuberculosis infectionin a colony of stumptail macaques (Macaca arctoides). J. Infect. Dis.155:1011-1019.

5. Eltholth, M. M., V. R. Marsh, W. S. Van, and F. J. Guitian. 2009.Contamination of food products with Mycobacterium aviumparatuberculosis: a systematic review. J. Appl. Microbiol.107:1061-1071. doi:JAM4286 [pii]; 10.1111/j.1365-2672.2009.04286.x[doi].

6. Golan, L., A. Livneh-Kol, E. Gonen, S. Yagel, I. Rosenshine, and N.Y. Shpigel. 2009. Mycobacterium avium paratuberculosis invades humansmall-intestinal goblet cells and elicits inflammation. J. Infect. Dis.199:350-354. doi:10.1086/596033 [doi].

7. Naser, S. A., G. Ghobrial, C. Romero, and J. F. Valentine. 2004.Culture of Mycobacterium avium subspecies paratuberculosis from theblood of patients with Crohn's disease. Lancet 364:1039-1044.

8. Bull, T. J., E. J. McMinn, K. Sidi-Boumedine, A. Skull, D. Durkin, P.Neild, G. Rhodes, R. Pickup, and J. Hermon-Taylor. 2003. Detection andverification of Mycobacterium avium subsp. paratuberculosis in freshileocolonic mucosal biopsy specimens from individuals with and withoutCrohn's disease. J. Clin. Microbiol. 41:2915-2923.

9. Naser, S. A., S. Thanigachalam, C. T. Dow, and M. T. Collins. 2013.Exploring the role of Mycobacterium avium subspecies paratuberculosis inthe pathogenesis of type 1 diabetes mellitus: a pilot study. Gut Pathog.5:14. doi:1757-4749-5-14 [pii]; 10.1186/1757-4749-5-14 [doi].

10. Masala, S., D. Paccagnini, D. Cossu, V. Brezar, A. Pacifico, N.Ahmed, R. Mallone, and L. A. Sechi. 2011. Antibodies recognizingMycobacterium avium paratuberculosis epitopes cross-react with thebeta-cell antigen ZnT8 in Sardinian type 1 diabetic patients. PLoS. One.6:e26931. doi:10.1371/journal.pone.0026931 [doi]; PONE-D-11-14979 [pii].

11. Lombard, J. E., I. A. Gardner, S. R. Jafarzadeh, C. P. Fossler, B.Harris, R. T. Capsel, B. A. Wagner, and W. 0. Johnson. 2013. Herd-levelprevalence of Mycobacterium avium subsp. paratuberculosis infection inUnited States dairy herds in 2007. Prev. Vet. Med. 108:234-238.doi:S0167-5877(12)00267-X [pii]; 10.1016/j.prevetmed.2012.08.006 [doi].

12. Hermon-Taylor, J. 2009. Mycobacterium avium subspeciesparatuberculosis, Crohn's disease and the Doomsday scenario. Gut Pathog.1:15. doi:1757-4749-1-15 [pii]; 10.1186/1757-4749-1-15 [doi].

13. Lei, L., B. L. Plattner, and J. M. Hostetter. 2008. LiveMycobacterium avium subsp. paratuberculosis and a killed-bacteriumvaccine induce distinct subcutaneous granulomas, with unique cellularand cytokine profiles. Clin. Vaccine Immunol. 15:783-793.doi:CVI.00480-07 [pii]; 10.1128/CVI.00480-07 [doi].

14. Losinger, W. C. 2005. Economic impact of reduced milk productionassociated with Johne's disease on dairy operations in the USA. Journalof Dairy Research 72:425-432.

15. Uzonna, J. E., P. Chilton, R. H. Whitlock, P. L. Habecker, P. Scott,and R. W. Sweeney. 2003. Efficacy of commercial and field-strainMycobacterium paratuberculosis vaccinations with recombinant IL-12 in abovine experimental infection model. Vaccine 21:3101-3109.doi:S0264410X03002615 [pii].

16. Kalis, C. H., J. W. Hesselink, H. W. Barkema, and M. T. Collins.2001. Use of long-term vaccination with a killed vaccine to preventfecal shedding of Mycobacterium avium subsp paratuberculosis in dairyherds. Am. J. Vet. Res. 62:270-274.

17. Patterson, C. J., M. LaVenture, S. S. Hurley, and J. P. Davis. 1988.Accidental self-inoculation with Mycobacterium paratuberculosis bacterin(Johne's bacterin) by veterinarians in Wisconsin. J. Am. Vet. Med.Assoc. 192:1197-1199.

18. Cossu, A., L. A. Sechi, S. Zanetti, and V. Rosu. 2012. Geneexpression profiling of Mycobacterium avium subsp. paratuberculosis insimulated multi-stress conditions and within THP-1 cells reveals a newkind of interactive intramacrophage behaviour. BMC. Microbiol. 12:87.doi:1471-2180-12-87 [pii]; 10.1186/1471-2180-12-87 [doi].

19. Ghosh, P., C. W. Wu, and A. M. Talaat. 2013. Key Role for theAlternative Sigma Factor, SigH, in the Intracellular Life ofMycobacterium avium subsp. paratuberculosis during Macrophage Stress.Infect. Immun. 81:2242-2257.

20. Li, L., J. P. Bannantine, Q. Zhang, A. Amonsin, B. J. May, D. Alt,N. Banerji, S. Kanjilal, and V. Kapur. 2005. The complete genomesequence of Mycobacterium avium subspecies paratuberculosis. Proc. Natl.Acad. Sci U.S.A 102:12344-12349.

21. Janagama, H. K., E. A. Lamont, S. George, J. P. Bannantine, W. W.Xu, Z. J. Tu, S. J. Wells, J. Schefers, and S. Sreevatsan. 2010. Primarytranscriptomes of Mycobacterium avium subsp. paratuberculosis revealproprietary pathways in tissue and macrophages. BMC Genomics 11:561.

22. Schnappinger, D., S. Ehrt, M. I. Voskuil, Y. Liu, J. A. Mangan, I.M. Monahan, G. Dolganov, B. Efron, P. D. Butcher, C. Nathan, and G. K.Schoolnik. 2003. Transcriptional Adaptation of Mycobacteriumtuberculosis within Macrophages: Insights into the PhagosomalEnvironment. J. Exp. Med. 198:693-704. doi:10.1084/jem.20030846 [doi];jem.20030846 [pii].

23. Cole, S. T., R. Brosch, J. Parkhill, T. Gamier, C. Churcher, D.Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry, III, F. Tekaia,K. Badcock, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. Davies,K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby,K. Jagels, A. Krogh, J. McLean, S. Moule, L. Murphy, K. Oliver, J.Osborne, M. A. Quail, M. A. Rajandream, J. Rogers, S. Rutter, K. Seeger,J. Skelton, R. Squares, S. Squares, J. E. Sulston, K. Taylor, S.Whitehead, and B. G. Barrell. 1998. Deciphering the biology ofMycobacterium tuberculosis from the complete genome sequence. Nature393:537-544. doi:10.1038/31159 [doi].

24. Hahn, M. Y., S. Raman, M. Anaya, and R. N. Husson. 2005. Themycobacterium tuberculosis extracytoplasmic-function sigma factor SigLregulates polyketide synthases and secreted or membrane proteins and isrequired for virulence. Journal of Bacteriology 187:7062-7071.

25. Pelicic, V., M. Jackson, J. M. Reyrat, W. R. Jacobs, Jr., B.Gicquel, and C. Guilhot. 1997. Efficient allelic exchange and transposonmutagenesis in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A94:10955-10960.

26. Ward, S. K., B. Abomoelak, E. A. Hoye, H. Steinberg, and A. M.Talaat. 2010. CtpV: a putative copper exporter required for fullvirulence of Mycobacterium tuberculosis. Mol. Microbiol. 77:1096-1110.doi:MM17273 [pii]; 10.1111/j.1365-2958.2010.07273.x [doi].

27. Kaushal, D., B. G. Schroeder, S. Tyagi, T. Yoshimatsu, C. Scott, C.Ko, L. Carpenter, J. Mehrotra, Y. C. Manabe, R. D. Fleischmann, and W.R. Bishai. 2002. Reduced immunopathology and mortality despite tissuepersistence in a Mycobacterium tuberculosis mutant lacking alternativesigma factor, SigH. Proc. Natl. Acad. Sci U.S.A 99:8330-8335.

28. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J.Zajac, J. D. Miller, J. Slansky, and R. Ahmed. 1998. Countingantigen-specific CD8 T cells: a reevaluation of bystander activationduring viral infection. Immunity. 8:177-187. doi:S1074-7613 (00)80470-7[pii].

29. Frey, A., C. J. Di, and D. Zurakowski. 1998. A statistically definedendpoint titer determination method for immunoassays. J. Immunol.Methods 221:35-41. doi:S0022-1759 (98)00170-7 [pii].

30. Gilliland, S. E., T. E. Staley, and L. J. Bush. 1984. Importance ofbile tolerance of Lactobacillus acidophilus used as a dietary adjunct.J. Dairy Sci. 67:3045-3051. doi:50022-0302 (84)81670-7 [pii];10.3168/jds.S0022-0302(84)81670-7 [doi].

31. Sung, N. and M. T. Collins. 2003. Variation in resistance ofMycobacterium paratuberculosis to acid environments as a function ofculture medium. Appl. Environ. Microbiol. 69:6833-6840.

32. Dainese, E., S. Rodrigue, G. Delogu, R. Provvedi, L. Laflamme, R.Brzezinski, G. Fadda, I. Smith, L. Gaudreau, G. Palu, and R. Manganelli.2006. Posttranslational regulation of Mycobacterium tuberculosisextracytoplasmic-function sigma factor sigma(L) and roles in virulenceand in global regulation of gene expression. Infect. Immun.74:2457-2461.

33. Khader, S. A. and R. Gopal. 2010. IL-17 in protective immunity tointracellular pathogens. Virulence. 1:423-427. doi:12862 [pii];10.4161/viru.1.5.12862 [doi].

34. Shin, S. J., C.-W. Wu, H. Steinberg, and A. M. Talaat. 2006.Identification of Novel Virulence Determinants in Mycobacteriumparatuberculosis by Screening a Library of Insertional Mutants. Infec.Immun. 7:3825-3833.

35. Begg, D. J. and J. F. Griffin. 2005. Vaccination of sheep against M.paratuberculosis: immune parameters and protective efficacy. Vaccine23:4999-5008. doi:S0264-410 X(05)00563-3 [pii];10.1016/j.vaccine.2005.05.031 [doi].

36. Stabel, J. R. and S. Robbe-Austerman. 2011. Early immune markersassociated with Mycobacterium avium subsp. paratuberculosis infection ina neonatal calf model. Clin. Vaccine Immunol. 18:393-405.doi:CVI.00359-10 [pii]; 10.1128/CVI.00359-10 [doi].

37. Wu, C. W., S. K. Schmoller, S. J. Shin, and A. M. Talaat. 2007.Defining the stressome of Mycobacterium avium subsp paratuberculosis invitro and in naturally infected cows. Journal of Bacteriology189:7877-7886.

38. Gunn, J. S. 2000. Mechanisms of bacterial resistance and response tobile. Microbes. Infect. 2:907-913. doi:S1286-4579 (00)00392-0 [pii].

39. Prieto, A. I., F. Ramos-Morales, and J. Casadesus. 2006. Repair ofDNA damage induced by bile salts in Salmonella enterica. Genetics174:575-584. doi:genetics.106.060889 [pii]; 10.1534/genetics.106.060889[doi].

40. den Hengst, C. D. and M. J. Buttner. 2008. Redox control inactinobacteria. Biochim. Biophys. Acta. 1780:1201-1216.doi:S0304-4165(08)00009-3 [pii]; 10.1016/j.bbagen.2008.01.008 [doi].

41. Coynault, C., V. Robbe-Saule, and F. Norel. 1996. Virulence andvaccine potential of Salmonella typhimurium mutants deficient in theexpression of the RpoS (sigma S) regulon. Mol. Microbiol. 22:149-160.

42. Hernandez, P. R., L. D. Aguilar, I. Smith, and R. Manganelli. 2010.Immunogenicity and protection induced by a Mycobacterium tuberculosissigE mutant in a BALB/c mouse model of progressive pulmonarytuberculosis. Infect. Immun. 78:3168-3176. doi:IAI.00023-10 [pii];10.1128/IAI.00023-10 [doi].

43. Park, K. T., A. J. Allen, J. P. Bannantine, K. S. Seo, M. J.Hamilton, G. S. Abdellrazeq, H. M. Rihan, A. Grimm, and W. C. Davis.2011. Evaluation of two mutants of Mycobacterium avium subsp.paratuberculosis as candidates for a live attenuated vaccine for Johne'sdisease. Vaccine 29:4709-4719. doi:S0264-410 X(11)00645-1 [pii];10.1016/j.vaccine.2011.04.090 [doi].

44. Chen, J. W., S. M. Faisal, S. Chandra, S. P. McDonough, M. A.Moreira, J. Scaria, C. F. Chang, J. P. Bannantine, B. Akey, and Y. F.Chang. 2012. Immunogenicity and protective efficacy of the Mycobacteriumavium subsp. paratuberculosis attenuated mutants against challenge in amouse model. Vaccine. 30:3015-3025. doi:S0264-410 X(11)01798-1 [pii];10.1016/j.vaccine.2011.11.029 [doi].

45. O'Garra, A., P. S. Redford, F. W. McNab, C. I. Bloom, R. J.Wilkinson, and M. P. Berry. 2013. The immune response in tuberculosis.Annu. Rev. Immunol. 31:475-527.doi:10.1146/annurev-immunol-032712-095939 [doi].

46. Begg, D. J., S. K. de, N. Carter, K. M. Plain, A. Purdie, and R. J.Whittington. 2011. Does a Th1 over Th2 dominancy really exist in theearly stages of Mycobacterium avium subspecies paratuberculosisinfections? Immunobiology 216:840-846. doi:S0171-2985 (10)00220-2 [pii];10.1016/j.imbio.2010.12.004 [doi].

47. Scandurra, G. M., G. W. de Lisle, S. M. Cavaignac, M. Young, R. P.Kawakami, and D. M. Collins. 2010. Assessment of live candidate vaccinesfor paratuberculosis in animal models and macrophages. Infect. Immun.78:1383-1389. doi:IAI.01020-09 [pii]; 10.1128/IAI.01020-09 [doi].

48. Jeyanathan, M., D. C. Alexander, C. Y. Turenne, C. Girard, and M. A.Behr. 2006. Evaluation of in situ methods used to detect Mycobacteriumavium subsp. paratuberculosis in samples from patients with Crohn'sdisease. J. Clin. Microbiol. 44:2942-2950. doi:44/8/2942 [pii];10.1128/JCM.00585-06 [doi].

49. Seiler, P., T. Ulrichs, S. Bandermann, L. Pradl, S. Jorg, V. Krenn,L. Morawietz, S. H. Kaufmann, and P. Aichele. 2003. Cell-wallalterations as an attribute of Mycobacterium tuberculosis in latentinfection. J. Infect. Dis. 188:1326-1331. doi:JID30430 [pii];10.1086/378563 [doi].

1. A method of differentiating between naïve subjects and subjects thathave been vaccinated with a mycobacterium vaccine, the method comprisingthe steps of (a) obtaining a sample from the subject; and (b) measuringINF-γ and anti-PPDj IgG levels in the sample, wherein increased levelsof INF-γ and anti-PPDj IgG as compared to levels in a known naïvesubject indicate a subject has been vaccinated.
 2. The method of claim1, wherein the mycobacterium vaccine comprises a mycobacterium mutantcomprising at least partial deletion of at least one gene sequenceencoding global gene regulators (GGRs) selected from the groupconsisting of sigH, sigL, sigE, sigB, and ECF-1, wherein the at leastpartial deletion includes insertion of a recombinant sequence byhomologous recombination into the coding region of the GGR.
 3. Themethod of claim 1, wherein measuring INF-γ and anti-PPDj IgG levels isdone using ELISA and horseradish peroxidase conjugated rabbit anti-mouseantibody.
 4. The method of claim 2, additionally comprising the step ofamplifying the recombinant sequence using primers specific for therecombinant sequence to confirm the vaccination status of the subject.5. The method of claim 4, wherein the amplification is loop-mediatedisothermal amplification (LAMP).
 6. The method of claim 1, wherein boththe naïve and the vaccinated subject do not have detectable levels ofIL-17A.
 7. The method of claim 1, wherein the biological sample isselected from the group consisting of saliva, sputum, blood, plasma,serum, urine, feces, cerebrospinal fluid, amniotic fluid, wound exudate,or tissue of the subject.
 8. The method of claim 2, wherein themycobacterium mutant is selected from the group consisting ofMycobacterium avium subspecies paratuberculosis (M. ap), Mycobacteriumbovis (M. bovis), Mycobacterium tuberculosis (M. tuberculosis), andmixtures thereof.
 9. The method of claim 8, wherein the mycobacteriummutant is M. ap.
 10. The method of claim 8, wherein the mycobacteriummutant is M. bovis.
 11. The method of claim 8, wherein the mycobacteriummutant is M. tuberculosis.
 12. The method of claim 2, wherein the GGR isM. ap sigH (SEQ ID NO:1) or a sequence substantially identical to SEQ IDNO:1.
 13. The method of claim 2, wherein the GGR is M. ap sigL (SEQ IDNO:2) or a sequence substantially identical to SEQ ID NO:2.
 14. Themethod of claim 2, wherein the GGR is M. ap sigE (SEQ ID NO:3) or asequence substantially identical to SEQ ID NO:3.
 15. The method of claim2, wherein the GGR is M. ap ECF-1 (SEQ ID NO:4) or a sequencesubstantially identical to SEQ ID NO:4.